19 research outputs found

    Substrate Specificity of SAMHD1 Triphosphohydrolase Activity Is Controlled by Deoxyribonucleoside Triphosphates and Phosphorylation at Thr592

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    The sterile alpha motif (SAM) and histidine-aspartate (HD) domain containing protein 1 (SAMHD1) constitute a triphosphohydrolase that converts deoxyribonucleoside triphosphates (dNTPs) into deoxyribonucleosides and triphosphates. SAMHD1 exists in multiple states. The monomer and apo- or GTP-bound dimer are catalytically inactive. Binding of dNTP at allosteric site 2 (AS2), adjacent to GTP-binding allosteric site 1 (AS1), induces formation of the tetramer, the catalytically active form. We have developed an enzyme kinetic assay, tailored to control specific dNTP binding at each site, allowing us to determine the kinetic binding parameters of individual dNTPs at both the AS2 and catalytic sites for all possible combinations of dNTP binding at both sites. Here, we show that the apparent <i>K</i><sub>m</sub> values of dNTPs at AS2 vary in the order of dCTP < dGTP < dATP < dTTP. Interestingly, dCTP binding at AS2 significantly reduces the dCTP hydrolysis rate, which is restored to a rate comparable to that of other dNTPs upon dGTP, dATP, or dTTP binding at AS2. Strikingly, a phosphomimetic mutant, Thr592Asp SAMHD1 as well as phospho-Thr592, show a significantly altered substrate specificity, with the rate of dCTP hydrolysis being selectively reduced regardless of which dNTP binds at AS2. Furthermore, cyclin A2 binding at the C-terminus of SAMHD1 induces the disassembly of the SAMHD1 tetramer, suggesting an additional layer of SAMHD1 activity modulation by cyclin A2/CDK2 kinase. Together, our results reveal multiple allosteric mechanisms for controlling the rate of dNTP destruction by SAMHD1

    <sup>1</sup>H–<sup>13</sup>C/<sup>1</sup>H–<sup>15</sup>N Heteronuclear Dipolar Recoupling by R-Symmetry Sequences Under Fast Magic Angle Spinning for Dynamics Analysis of Biological and Organic Solids

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    Fast magic angle spinning (MAS) NMR spectroscopy is becoming increasingly important in structural and dynamics studies of biological systems and inorganic materials. Superior spectral resolution due to the efficient averaging of the dipolar couplings can be attained at MAS frequencies of 40 kHz and higher with appropriate decoupling techniques, while proton detection gives rise to significant sensitivity gains, therefore making fast MAS conditions advantageous across the board compared with the conventional slow- and moderate-MAS approaches. At the same time, many of the dipolar recoupling approaches that currently constitute the basis for structural and dynamics studies of solid materials and that are designed for MAS frequencies of 20 kHz and below, fail above 30 kHz. In this report, we present an approach for <sup>1</sup>H–<sup>13</sup>C/<sup>1</sup>H–<sup>15</sup>N heteronuclear dipolar recoupling under fast MAS conditions using R-type symmetry sequences, which is suitable even for fully protonated systems. A series of rotor-synchronized R-type symmetry pulse schemes are explored for the determination of structure and dynamics in biological and organic systems. The investigations of the performance of the various R<i>N</i><sub><i>n</i></sub><sup><i>v</i></sup>-symmetry sequences at the MAS frequency of 40 kHz experimentally and by numerical simulations on [U-<sup>13</sup>C,<sup>15</sup>N]-alanine and [U-<sup>13</sup>C,<sup>15</sup>N]-<i>N</i>-acetyl-valine, revealed excellent performance for sequences with high symmetry number ratio (<i>N</i>/2<i>n</i> > 2.5). Further applications of this approach are presented for two proteins, sparsely <sup>13</sup>C/uniformly <sup>15</sup>N-enriched CAP-Gly domain of dynactin and U-<sup>13</sup>C,<sup>15</sup>N-Tyr enriched C-terminal domain of HIV-1 CA protein. Two-dimensional (2D) and 3D R16<sub>3</sub><sup>2</sup>-based DIPSHIFT experiments carried out at the MAS frequency of 40 kHz, yielded site-specific <sup>1</sup>H–<sup>13</sup>C/<sup>1</sup>H–<sup>15</sup>N heteronuclear dipolar coupling constants for CAP-Gly and CTD CA, reporting on the dynamic behavior of these proteins on time scales of nano- to microseconds. The R-symmetry-based dipolar recoupling under fast MAS is expected to find numerous applications in studies of protein assemblies and organic solids by MAS NMR spectroscopy

    SIVrcm Vpx utilizes a unique interface to degrade SAMHD1.

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    <p>(A) Alignment of the amino acids in Vpx that have been previously shown to be important for SIVmac Vpx-mediated degradation of SAMHD1, highlighted in in black with amino acid numbers above. (B) Wild type and mutant Vpx constructs were tested for their ability to degrade SAMHD1 by cotransfection in 293T cells, and analyzed by western blotting. HIV-2<sub>12 Mnd 17</sub> and Mac<sub>12 Mnd 17</sub> (or HIV-2<sub>12 RCM 17</sub> and Mac<sub>12 RCM 17</sub>) indicates the amino acids 12 through 17 of HIV-2 Vpx and SIVmac Vpx that have been changed to the corresponding amino acids found in SIVmnd2 Vpx (or SIVrcm Vpx), while RCM<sub>12 HIV-2 17</sub> and Mnd2<sub>12 HIV-2 17</sub> indicates that amino acids 12 through 17 of SIVrcm and SIVmnd2 Vpx have been changed to the corresponding amino acids found in HIV-2/SIVmac Vpx. – indicates no Vpx empty vector control.</p

    Vpx and Vpr have toggled throughout evolution in their requirement for the N- or the C-terminus of SAMHD1.

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    <p>(A) 293T cells were transfected with HA-SAMHD1, either WT or ΔC (left panel), or WT or chimeric Human-Rhesus SAMHD1 (consisting of residues 1–114 of human SAMHD1 and 115–626 of rhesus SAMHD1, right panels), plus or minus FLAG-Vpr, and degradation was measured by western blotting as described in the legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g001" target="_blank">Figure 1</a>. Rhesus SAMHD1 was used as all Vpr tested herein can degrade this SAMHD1. Human SAMHD1 was used in N-terminal chimeras as it contains multiple non-synonymous changes compared to other primate SAMHD1. (B) <i>In vitro</i> ubiquitylation of WT, ΔC, ΔN, and ΔN/ΔC DeBrazza's SAMHD1 (consisting of amino acids 115–595, schematic representation in top panels), in the presence or absence of SIVdeb Vpr, SIVmac Vpx, or SIVrcm Vpx. CRL4 and CRL4-DCAF1 alone were used as controls. Experiment preformed as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g004" target="_blank">Figure 4B</a> and ubiquitylation of each SAMHD1 construct was analyzed by western blotting. Timepoints shown are 0, 15, 30, and 60 minute incubations. (C) Schematic Vpx/Vpr phylogenetic tree as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g001" target="_blank">Figure 1A</a> and SAMHD1 diagrams depicting dependence of Vpx/Vpr on the N- or C-terminus of its autologous SAMHD1. Red indicates strong dependence based on all assays. Light pink indicates slight dependence based on co-IP data. Magenta indicates intermediate dependence.</p

    HIV-2 and SIVmac Vpx require the C-terminus of SAMHD1 for degradation.

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    <p>(A) Schematic Vpx/Vpr phylogenetic tree based on <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat.1003496-Lim1" target="_blank">[21]</a> of select lentiviral Vpx and Vpr proteins that degrade SAMHD1. (B) Schematic representation of SAMHD1 (WT) and C-terminal truncation (ΔC), including the SAM and HD domains shown in grey. The deletion at the C-terminus of SAMHD1 (green dotted line) truncates the protein at amino acid 611. Amino acid numbers are shown to indicate the relative boundaries of the domains. (C) 293T cells were transfected with HA-SAMHD1, either WT or ΔC (green), plus or minus (−) FLAG-Vpx from autologous viruses, and degradation was measured by western blotting. SAMHD1 levels are shown on the top blot, Vpx levels are shown in the middle blot, and tubulin levels are shown in the bottom blot (as a loading control). Note the differences between degradation of the WT SAMHD1 versus the ΔC SAMHD1 in the presence of some Vpx proteins, but not others.</p

    Vpx specificity for N- versus C-terminal binding of SAMHD1 is independent of the SAMHD1 homologue.

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    <p>(A) Empty vector control, C-terminal binding FLAG-SIVmac Vpx, or N-terminal binding FLAG-SIVmnd2 Vpx were co-transfected in 293T cells with HA-SAMHD1, either WT or ΔC, from multiple primate species, and degradation of SAMHD1 was measured by western blotting. (B) Same as in (A) except chimeric SAMHD1 proteins were used instead of WT or ΔC.</p

    N- and C-terminal binding Vpx proteins degrade SAMHD1 through a conserved mechanism.

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    <p>(A) Schematic representation of wild type (WT) SAMHD1, C-terminally truncated SAMHD1 (ΔC, shown in green), and N-terminally truncated SAMHD1 (ΔN, shown in blue). Amino acid numbers of the truncations are shown, with dotted line indicating the truncated region of SAMHD1. (B) HA-SAMHD1 (WT, ΔC, and ΔN) were transiently co-expressed in 293T cells with the autologous FLAG-Vpx and immunoprecipitated from whole cell extracts with anti-HA resin. HA-SAMHD1, FLAG-Vpx, DCAF, and DDB1 were detected in immune complexes (top panels) or extracts (bottom panels) by western blotting. * denotes the antibody light-chain. (C) <i>In vitro</i> ubiquitylation of WT, ΔC, and ΔN rhesus SAMHD1, in the presence or absence of SIVmac Vpx (left panel) or SIVrcm Vpx (right panel). SAMHD1 and Vpx were incubated with Cul4, DCAF1c, RBX1, UBA1 (an E1 enzyme), UbcH5b (an E2 enzyme) and FLAG-tagged ubiquitin for increasing time (0, 15, 30 min), and ubiquitylation of each SAMHD1 construct was analyzed by western blotting.</p

    SIVmnd2 and SIVrcm Vpx require the N-terminus of SAMHD1 for degradation.

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    <p>(A) Schematic representation of WT SAMHD1 and chimeric proteins. Amino acids in the N-terminus that differ between human (Hu) SAMHD1 and mandrill (Mnd) or RCM SAMHD1 are shown as tick-marks, while codons that differ and have been shown to be evolving under strong positive selection (amino acids 32, 36, and 107) are shown with arrowheads. The regions of the chimeric SAMHD1 protein coming from the human gene are shown in purple, while the regions coming from mandrill or RCM SAMHD1 are shown in grey. (B) 293T cells were transfected with HA-tagged WT or chimeric SAMHD1, plus or minus (−) FLAG-Vpx, and degradation was measured by western blotting as described in the legend for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003496#ppat-1003496-g001" target="_blank">Figure 1</a>.</p

    Straightforward Delivery of Linearized Double-Stranded DNA Encoding sgRNA and Donor DNA for the Generation of Single Nucleotide Variants Based on the CRISPR/Cas9 System

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    CRISPR/Cas9 for genome editing requires delivery of a guide RNA sequence and donor DNA for targeted homologous recombination. Typically, single-stranded oligodeoxynucleotide, serving as the donor template, and a plasmid encoding guide RNA are delivered as two separate components. However, in the multiplexed generation of single nucleotide variants, this two-component delivery system is limited by difficulty of delivering a matched pair of sgRNA and donor DNA to the target cell. Here, we describe a novel codelivery system called “sgR-DNA” that uses a linearized double-stranded DNA consisting of donor DNA component and a component encoding sgRNA. Our sgR-DNA-based method is simple to implement because it does not require cloning steps. We also report the potential of our delivery system to generate multiplex genomic substitutions in <i>Escherichia coli</i> and human cells

    Straightforward Delivery of Linearized Double-Stranded DNA Encoding sgRNA and Donor DNA for the Generation of Single Nucleotide Variants Based on the CRISPR/Cas9 System

    No full text
    CRISPR/Cas9 for genome editing requires delivery of a guide RNA sequence and donor DNA for targeted homologous recombination. Typically, single-stranded oligodeoxynucleotide, serving as the donor template, and a plasmid encoding guide RNA are delivered as two separate components. However, in the multiplexed generation of single nucleotide variants, this two-component delivery system is limited by difficulty of delivering a matched pair of sgRNA and donor DNA to the target cell. Here, we describe a novel codelivery system called “sgR-DNA” that uses a linearized double-stranded DNA consisting of donor DNA component and a component encoding sgRNA. Our sgR-DNA-based method is simple to implement because it does not require cloning steps. We also report the potential of our delivery system to generate multiplex genomic substitutions in <i>Escherichia coli</i> and human cells
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