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Mutagenesis, Site-Directed (1):
Recent article
Mutagenesis, Site-Directed
Abstract
We have discovered that other cellular stressors are integrated with glucocorticoid signaling through a new hormone-independent phosphorylation site, Ser134, on the human glucocorticoid receptor (GR). Ser134 phosphorylation is induced by a variety of stress-activating stimuli in a p38 mitogen-activa...
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PMID: 21930780
PDF is available here.
PDF is available here.
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Abstract
We have recently shown that some viruses carrying capsid (CA) sequences from clinical isolates can be more sensitive to this restriction factor. In this study we evaluated the contribution to this phenotype of CA mutations known to be associated with escape from cytotoxic T lymphocyte (CTL) response...
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PMID: 21917976
PDF is available here.
PDF is available here.
Abstract
We developed a nuclear magnetic resonance approach that enabled direct detection of structural elements within the intact leader (712-nucleotide dimer) that are critical for genome packaging. Residues spanning the gag start codon (AUG) form a hairpin in the monomeric leader and base pair with residu...
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PMID: 21998393
PDF is available here.
PDF is available here.
Abstract
We determined the high-resolution crystal structures of the EV71 3C(pro)/rupintrivir complex, showing that although rupintrivir interacts with EV71 3C(pro) similarly to HRV 3C(pro), the C terminus of the inhibitor cannot accommodate the leaving-group pockets of EV71 3C(pro). Our structures reveal th...
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PMID: 21813612
PDF is available here.
PDF is available here.
Abstract
We have studied the composition of complexes that copurify with Spb4 tagged by tandem affinity purification (TAP-tagged Spb4). These complexes contain mainly the 27SB pre-rRNAs and about 50 ribosome biogenesis proteins, primarily components of early pre-60S ribosomal particles. To a lesser extent, s...
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PMID: 21825077
PDF is available here.
PDF is available here.
Journal of Biological Chemistry (286)39 2011
Abstract
We have previously shown that two of the polyphenols from green tea (epigallocatechin gallate (EGCG) and epicatechin gallate (ECG)) inhibit GDH in vitro and that EGCG blocks GDH-mediated insulin secretion in wild type rat islets. Using structural and site-directed mutagenesis studies, we demonstrate...
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PMID: 21813650
PDF is available here.
PDF is available here.
Abstract
We report that the histone acetyltransferase (HAT), p300, specifically acetylates BTas both in vivo and in vitro. Further studies demonstrated that BTas acetylation markedly enhances its transactivation activity. Mutagenesis analysis identified three lysines at positions 66, 109 and 110 in BTas that...
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PMID: 21813148
PDF is available here.
PDF is available here.
Abstract
We developed a program to parse sequences according to predicted topology and then analyze topologically related regions. This approach identified three conserved motifs that served as the basis for subsequent mutagenesis and functional studies. This work reveals that several inter-transmembrane loo...
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PMID: 21812456
PDF is available here.
PDF is available here.
Abstract
We have solved the crystal structure of the RNA recognition and leucine-rich repeat motifs of TAP bound to one symmetrical half of the CTE RNA. L-shaped conformations of protein and RNA are involved in a mutual molecular embrace on complex formation. We have monitored the impact of structure-guided...
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PMID: 21822283
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1808)10 2011
Abstract
Replacement of the glycine at position 117 by a cysteine in the melibiose permease creates an interesting phenotype: while the mutant transporter shows still transport activity comparable to the wild type its pre steady-state kinetic properties are drastically altered. The transient charge displaceme...
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PMID: 21801712
PDF is available here.
PDF is available here.
Abstract
These results identify the MinD-dependent conformational changes in MinE that convert it from a latent to an active form and lead to a model of how MinE persists at the MinD-membrane surface. PAPERFLICK:...
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PMID: 21816275
PDF is available here.
PDF is available here.
Abstract
We show that a previously uncharacterized member of the I-BAR subfamily, Pinkbar, is specifically expressed in intestinal epithelial cells, where it localizes to Rab13-positive vesicles and to the plasma membrane at intercellular junctions. Notably, the BAR domain of Pinkbar does not induce membrane...
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PMID: 21743456
PDF is available here.
PDF is available here.
Abstract
To study the protocol of construction of a KCNQ2-c.812G>T mutant and it's eukaryotic expression vector, the c.812G>T (p.G271V) mutation which was detected in a Chinese pedigree of benign familial infantile convulsions, and to examine the expression of mutant protein in human embyonic kidney (HEK) 293...
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PMID: 21849107
PDF is available here.
PDF is available here.
Abstract
We determined the high-resolution x-ray crystal structure of the GspB binding region (GspB(BR)), both alone and in complex with a disaccharide precursor to sialyl-T antigen. Analysis of the GspB(BR) structure revealed that it is comprised of three independently folded subdomains or modules: 1) an Ig...
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PMID: 21765814
PDF is available here.
PDF is available here.
Abstract
We present the crystal structures of the SECRET domain of vTNFR CrmD encoded by ectromelia virus and its complex with chemokine CX3CL1. The SECRET domain adopts a β-sandwich fold and utilizes its β-sheet I surface to interact with CX3CL1, representing a new chemokine-binding manner of viral CKBPs....
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PMID: 21829356
PDF is available here.
PDF is available here.
Abstract
We show that FrsA converts pyruvate to acetaldehyde and carbon dioxide in a cofactor-independent manner and that its pyruvate decarboxylation activity is enhanced by the dephosphorylated form of IIA(Glc) (d-IIA(Glc)). Crystal structures of FrsA and its complex with d-IIA(Glc) revealed residues requi...
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PMID: 21623357
PDF is available here.
PDF is available here.
Abstract
We also predict that Glu15, Lys43, and Tyr46 may be important in the catalytic efficiency and thermostability of enzyme. The new structural and mechanistic insights obtained from computational study should be valuable for detailed structures and mechanisms of the member of the DJ-1 superfamily....
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PMID: 20711794
PDF is available here.
PDF is available here.
Abstract
We identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and...
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PMID: 21722859
PDF is available here.
PDF is available here.
Abstract
We have recently shown that an octopamine-activated G-protein-coupled receptor (GPCR; DmOctα1Rb, CG3856) from Drosophila initiates Ca(2+) oscillations. Here, we have unraveled the molecular basis of cellular Ca(2+) signaling controlled by the DmOctα1Rb receptor using a combination of pharmacologic...
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PMID: 21478261
PDF is available here.
PDF is available here.
Abstract
Our results showed that the hSMVT protein is glycosylated and that this glycosylation is important for its function. Studies utilizing site-directed mutagenesis revealed that the N-glycosylation sites at positions Asn(138) and Asn(489) are important for the function of hSMVT and that mutating these...
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PMID: 21570947
PDF is available here.
PDF is available here.
Abstract
We found that hKir2.1 current was reversibly inhibited by the broad spectrum PTK inhibitor genistein and the highly selective EGFR (epidermal growth factor receptor) kinase inhibitor AG556 in a concentration-dependent manner. The inhibition of hKir2.1 channels by genistein or AG556 was countered by...
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PMID: 21570948
PDF is available here.
PDF is available here.
Abstract
We undertook a phenotypic and biochemical study of the flax nucleotide binding (NB)-ARC leucine-rich repeat protein, M. Using Agrobacterium-mediated transient expression in flax cotyledons, site-directed mutations of key residues within the P-loop, kinase 2, and MHD motifs within the NB-ARC domain o...
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PMID: 21539434
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1814)7 2011
Abstract
The plasma phospholipid transfer protein (PLTP) plays a key role in lipid and lipoprotein metabolism. It has six potential N-glycosylation sites. To study the impact of these sites on PLTP secretion and activity, six variants containing serine to alanine point mutations were prepared by site-directed...
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PMID: 21515415
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1814)7 2011
Abstract
We selected residues (42)E and (43)E and the aromatic triplet (82)YYY(84) for site-directed mutagenesis to study the interactions with Pcho and p-nitrophenylphosphate as substrates of PchP. Because mutations in (42)E, (43)E and the three tyrosine residues affect both the substrate affinity and the i...
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PMID: 21515416
PDF is available here.
PDF is available here.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology (27)4 2011
Abstract
To construct and express a trichosanthin(TCS)gene mutant and purify the expressed product.
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PMID: 21481314
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1808)9 2011
Abstract
We examined these and other critical residues by site-directed mutagenesis substituting Asn227→Ala, Asp, Arg; Ile233→Ala; Leu243→Ala; Glu247→Asp, Gln; Glu248→Asp, Gln. Mutant NHE1 proteins were characterized in AP-1 cells, which do not express endogenous NHE1. All the TMVI critical amino a...
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PMID: 21600870
PDF is available here.
PDF is available here.
Abstract
In human carbonic anhydrase II (HCA II), the mutation of position 64 from histidine to alanine (H64A) disrupts the rate limiting proton transfer (PT) event, resulting in a reduction of the catalytic activity of the enzyme as compared to the wild-type. Potential of mean force (PMF) calculations utiliz...
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PMID: 21452838
PDF is available here.
PDF is available here.
Biopolymers (95)10 2011
Abstract
As a serine/threonine protein kinase, glycogen synthase kinase 3β (GSK3β) is an essential component of several cellular processes, including insulin, growth factor, and Wnt signaling. The conserved K85 is important to GSK3β activity and FRATide binding. To elucidate the mechanisms concerning kinas...
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PMID: 21442609
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1808)7 2011
Abstract
We investigated the existence of such a motif in the BAX sequence. Residues 113 to 119 of the recombinant BAX α5-helix, LFYFASK, correspond with the sequence motif described for the consensus pattern, -L/V-(X)(1-5)-Y-(X)(1-5)-R/K. Functional characterization of the point mutations, K119A, Y115F, an...
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PMID: 21440528
PDF is available here.
PDF is available here.
Cell Host & Microbe (9)3 2011
Abstract
We determine the crystal structure of the L6 TIR domain and show that, although dispensable for pathogenic effector protein recognition, the TIR domain alone is both necessary and sufficient for L6 immune signaling. We demonstrate that the L6 TIR domain self-associates, most likely forming a homodi...
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PMID: 21402359
PDF is available here.
PDF is available here.
Abstract
Glutaredoxins (Grxs) play important roles in the redox system via reduced glutathione as a reductant. A TcmonoGrx cDNA (1039 bp, EU158772) encoding a putative monothiol Grx was cloned from Taiwanofungus camphorata (formerly named Antrodia camphorata). The deduced amino acid sequence is conserved amon...
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PMID: 21395221
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1808)7 2011
Abstract
We hypothesized that cpt-cAMP may eliminate the self-inhibition of human ENaC thereby open channels. Regulation of self-inhibition by this compound in oocytes was analyzed using the two-electrode voltage clamp and Ussing chamber setups. External cpt-cAMP stimulated human but not rat and murine αβ...
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PMID: 21419751
PDF is available here.
PDF is available here.
Abstract
The variations in glucosidic linkage specificity observed in products of different glucansucrases appear to be based on relatively small differences in amino acid sequences in their sugar-binding acceptor subsites. Various amino acid mutations near active sites of DSRBCB4 dextransucrase from Leuconos...
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PMID: 21391600
PDF is available here.
PDF is available here.
Current Biology (21)5 2011
Abstract
We found that yeast and mammalian formins bind actin monomers but that this activity requires their C-terminal DAD domains. Furthermore, we observed that the DAD works in concert with the FH2 to enhance nucleation without affecting the rate of filament elongation. We dissected this mechanism in mDia...
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PMID: 21333540
PDF is available here.
PDF is available here.
Abstract
To prepare and purify recombinant human NAMPT and NAMPT (H247A) proteins and to detect their enzymatic activity.
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PMID: 21488211
PDF is available here.
PDF is available here.
Abstract
To construct eukaryotic expression plasmids containing green fluorescent protein gene and CYP19 wild-type or its vatiants (W39R, R264C, W39R-R264C) and to observe its expression in MCF-7 and Bcap-37 cells.
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PMID: 21488216
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1814)5 2011
Abstract
We have analyzed 125 bacterial sequences with respect to sequence variations in this position revealing that in a small number of sequences alanine and methionine can be found. Here we report the detailed comparative biochemical analysis of three Cys-less variants of EF-Tu containing these substitut...
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PMID: 21338717
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1814)5 2011
Abstract
I formation is decreased between 3.5 and 5.4 orders of magnitude for the mutants compared to wild-type CcP, with the rate of the reaction between CcP(H52Q) and H(2)O(2) the slowest yet observed for any CcP mutant. A correlation between the rate of Compound I formation and the rate of HCN binding for...
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PMID: 21354339
PDF is available here.
PDF is available here.
Abstract
We show simultaneous binding of FH19 to C3b and FH20 to nonactivator surface glycosaminoglycans, and we show that both of these interactions are necessary for full binding of FH to C3b on nonactivator surfaces (i.e., for target discrimination). We also show that C3d could replace glycosaminoglycan b...
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PMID: 21285368
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1808)7 2011
Abstract
We have mutated the glutamines at 713 and 1375 to asparagine, methionine or leucine to determine the functional roles of these residues in Q-loops of MRP1. All these single mutants significantly decreased Mg·ATP binding and increased the K(m) (Mg·ATP) and V(max) values in Mg·ATP-dependent leukotr...
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PMID: 21315686
PDF is available here.
PDF is available here.
Abstract
We tested a series of cysteine-to-alanine mutants. Two of these mutants, C107A and C184A, showed no agonist-dependent activation in a cell-based reporter assay for mEP3γ, whereas none of the other cysteine-to-alanine mutations disrupted mEP3γ signal transduction. Total cell membranes prepared from...
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PMID: 21236356
PDF is available here.
PDF is available here.
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology (27)2 2011
Abstract
The results of luciferase assays revealed that overexpression of miR-196a could reduce the luciferase activity from the reporter construct containing the HOXA9 3'-UTR significantly. The activity of the reporter construct mutated at the specific miR-196a target site was unaffected. Protein and mRNA o...
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PMID: 21315047
PDF is available here.
PDF is available here.
Abstract
We analyzed the TP53 mutation status in 2435 consecutive CLL samples, including 1287 diagnostic samples and 1148 samples during follow-up, using FASAY (Functional Analysis of Separated Alleles in Yeast) and direct sequencing. In a cohort of 1287 diagnostic CLL samples, we identified 237 cases with T...
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PMID: 21232794
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1810)4 2011
Abstract
The glutathione transferase (GST) superfamily is involved in the detoxification of various xenobiotics. We have identified a GST mRNA that was induced in the fat bodies of a silkworm strain exhibiting diazinon resistance and have investigated the enzyme properties of this GST.
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PMID: 21237249
PDF is available here.
PDF is available here.
Biochimica et Biophysica Acta (1808)4 2011
Abstract
The human prostacyclin receptor (hIP) undergoes agonist-dependent trafficking involving a direct interaction with Rab11a GTPase. The region of interaction was localised to a 14 residue Rab11a binding domain (RBD) within the proximal carboxyl-terminal (C)-tail domain of the hIP, consisting of Val(299)...
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PMID: 21223948
PDF is available here.
PDF is available here.
Abstract
These results provided a basis for the development of h357 IgG as therapeutic use....
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PMID: 21305012
PDF is available here.
PDF is available here.
Abstract
We report the crystal structures of the Enterobacter sp. Px6-4 FADase and the enzyme in complex with substrate analogues. Our analyses revealed that FADase possessed a half-opened bottom β-barrel with the catalytic pocket located between the middle of the core β-barrel and the helical bottom. Its...
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PMID: 21283705
PDF is available here.
PDF is available here.
Abstract
From the studies reported here, we show that mutations of the Y- and LL-motifs, which effectively eliminate the amphipathic nature of the lytic peptide 2 (LLP2) domain or disrupt YW and LL motifs in a region spanning residues 795-803 (YWWNLLQYW), just C-terminal of LLP2, can dramatically interfere w...
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PMID: 21569545
PDF is available here.
PDF is available here.
Abstract
Both ornithine decarboxylase (ODC) and its regulatory protein, antizyme inhibitor (AZI), can bind with antizyme (AZ), but the latter has a higher AZ-binding affinity. The results of this study clearly identify the critical amino acid residues governing the difference in AZ-binding af...
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PMID: 21552531
PDF is available here.
PDF is available here.
Abstract
We demonstrated that the G1613A mutation in the HBV negative regulatory element (NRE) is a hotspot mutation in HCC patients. In this study, we further investigated the functional consequences of this mutation in the context of the full length HBV genome and its replication. We showed that the G1613A...
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PMID: 21814558
PDF is available here.
PDF is available here.
Abstract
We now demonstrate significant nuclear GR in the absence of ligand, which requires nuclear localisation signal 1 (NLS1). Live cell imaging reveals dramatic GR import into the nucleus through interphase and rapid exclusion of the GR from the nucleus at the onset of mitosis, which persists into early...
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PMID: 21829454
PDF is available here.
PDF is available here.
Abstract
We aimed to identify modifier genes that may contribute to the clinical course of Lafora disease patients with EPM2A or EPM2B mutations. We established a list of 43 genes coding for proteins related to laforin/malin function and/or glycogen metabolism and tested common polymorphisms for possible ass...
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PMID: 21738631
PDF is available here.
PDF is available here.
Abstract
We generated mutant HIV-2 viruses, each carrying one of the remaining 17 possible amino acid residues, and examined their sensitivity to CM TRIM5α-mediated restriction. Results showed that hydrophobic residues or those with ring structures were associated with sensitivity, while those with small si...
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PMID: 21829511
PDF is available here.
PDF is available here.
Abstract
We have determined atomic structures of two invertebrate homologs of PLCβ (PLC21) from cephalopod retina and identified a helix from the C-terminal regulatory region that interacts with a conserved surface of the catalytic core of the enzyme. Mutations designed to disrupt the analogous interaction...
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PMID: 21822282
PDF is available here.
PDF is available here.
Abstract
We found that zebrafish, but not human, Ngb can translocate into cells. Moreover, we demonstrated that a chimeric ZHHH Ngb protein, in which the module M1 of human Ngb is replaced by the corresponding region of zebrafish Ngb, can penetrate cell membranes and protect cells against oxidative stress-in...
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PMID: 21304818
PDF is available here.
PDF is available here.
Abstract
The Gag polyproteins play distinct roles during the replication cycle of retroviruses, hijacking many cellular machineries to fulfill them. In the case of the prototype foamy virus (PFV), Gag structural proteins undergo transient nuclear trafficking after their synthesis, returning back to the cytopl...
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PMID: 21255441
PDF is available here.
PDF is available here.
Abstract
We have accomplished thermodynamic analysis of mouse TREK-1 gating and functional testing of several deletion mutants. The predicted increase of the channel in-plane area (ΔA) of ~5 nm(2) in models was supported by the experimental ΔA of ~4 nm(2) derived from the slope of open probability versus m...
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PMID: 21057213
PDF is available here.
PDF is available here.
Thrombosis and Haemostasis (105)1 2011
Abstract
We investigated the role of cysteine residues in the CUB-1 domain in ADAMTS13 synthesis and activity. CUB-1 and cysteine mutations were expressed in mammalian cell lines and examined for synthesis, secretion, stability, and VWF-cleaving activity. When expressed as an isolated domain, CUB-1, but not...
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PMID: 20886194
PDF is available here.
PDF is available here.
Abstract
Our results support the model that the QS sRNAs bind to the 5' untranslated region of multiple mRNAs and alter the fate of one in a positive manner and several others in a negative manner. This mechanism ensures the proper timing of the QS response, which includes the expression of traits critical f...
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PMID: 21031313
PDF is available here.
PDF is available here.
Journal of Biological Chemistry (285)52 2010
Abstract
We used site-directed mutagenesis, UV spectroscopy, and x-ray crystallography to examine the catalytic role of Arg-104 and Arg-31. Exchange of Arg-104 with Ala, Ser, Thr, or Lys abolished 94.3-99.9% of the specific activity against LTA(4). Steady-state kinetics of R104A and R104S revealed that the K...
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PMID: 20980252
PDF is available here.
PDF is available here.