Data_Sheet_1_DRJAMM Is Involved in the Oxidative Resistance in Deinococcus radiodurans.docx

Proteins containing JAB1/MPN/MOV34 metalloenzyme (JAMM/MPN+) domains that have Zn2+-dependent deubiquitinase (DUB) activity are ubiquitous across among all domains of life. Recently, a homolog in Deinococcus radiodurans, DRJAMM, was reported to possess the ability to cleave DRMoaD-MoaE. However, the detailed biochemical characteristics of DRJAMM in vitro and its biological mechanism in vivo remain unclear. Here, we show that DRJAMM has an efficient in vitro catalytic activity in the presence of Mn2+, Ca2+, Mg2+, and Ni2+ in addition to the well-reported Zn2+, and strong adaptability at a wide range of temperatures. Disruption of drJAMM led to elevated sensitivity in response to H2O2in vivo compared to the wild-type R1. In particular, the expression level of MoaE, a product of DRJAMM cleavage, was also increased under H2O2 stress, indicating that DRJAMM is needed in the antioxidant process. Moreover, DRJAMM was also demonstrated to be necessary for dimethyl sulfoxide respiratory system in D. radiodurans. These data suggest that DRJAMM plays key roles in the process of oxidative resistance in D. radiodurans with multiple-choice of metal ions and temperatures.</p

Gold Nanoparticles Biosynthesized and Functionalized Using a Hydroxylated Tetraterpenoid Trigger Gene Expression Changes and Apoptosis in Cancer Cells

Understanding the synthetic mechanisms and cell–nanoparticle interactions of biosynthesized and functionalized gold nanoparticles (AuNPs) using natural products is of great importance for developing their applications in nanomedicine. In this study, we detailed the biotransformation mechanism of Au­(III) into AuNPs using a hydroxylated tetraterpenoid deinoxanthin (DX) from the extremophile Deinococcus radiodurans. During the process, Au­(III) was rapidly reduced to Au­(I) and subsequently reduced to Au(0) by deprotonation of the hydroxyl head groups of the tetraterpenoid. The oxidized form, deprotonated 2-ketodeinoxanthin (DX3), served as a surface-capping agent to stabilize the AuNPs. The functionalized DX–AuNPs demonstrated stronger inhibitory activity against cancer cells compared with sodium citrate–AuNPs and were nontoxic to normal cells. DX–AuNPs accumulated in the cytoplasm, organelles, and nuclei, and induced reactive oxygen species generation, DNA damage, and apoptosis within MCF-7 cancer cells. In the cells treated with DX–AuNPs, 374 genes, including RRAGC gene, were upregulated; 135 genes, including the genes encoding FOXM1 and NR4A1, were downregulated. These genes are mostly involved in metabolism, cell growth, DNA damage, oxidative stress, autophagy, and apoptosis. The anticancer activity of the DX–AuNPs was attributed to the alteration of gene expression and induction of apoptosis. Our results provide significant insight into the synthesis mechanism of AuNPs functionalized with natural tetraterpenoids, which possess enhanced anticancer potential

Mn(II) and Fe(II) modulate the binding activity of DR2539 <i>in vivo</i>.

<p>(A) Effects of divalent metals (50 µM) on expression of pRAZH in <i>D. radiodurans</i>. Data shown are the means ± standard deviations of three independent experiments. (B) Effects of Mn(II) (squares) and Fe(II) (circles) on the expression of pRAZH in wild-type samples expressing DR2539. (C) Effects of Mn(II) (squares) and Fe(II) (circles) on the expression of pRAZH in the <i>dr2539</i> null mutant. (D) Rea-time PCR analysis of the <i>dr1709</i> gene expression using <i>dr0089</i> as internal control gene. Longitudinal axes indicate the change fold of <i>dr1709</i> mRNA relative to controls. Control cells were cultured in medium without Mn(II). *, <i>P</i><0.05 relative to control. The data are the means ± standard deviations of three independent experiments.</p

Sequence alignment of the metal binding sites of DR2539 with other DxtR/MntR family members.

<p>ScaR (<i>Streptococcus gordonii</i>), SloR (<i>Streptococcus suis</i>), LCAS (<i>Lactobacillus casei</i>), SirR (<i>Corynebacterium glutamicum</i>), TroR (<i>Treponema pallidum</i>), TroR (<i>Treponema denticola</i>), IdeR (<i>Mycobacterium tuberculosis</i>), DtxR (<i>Corynebacterium diptheriae</i>), DR2539 (<i>Deinococcus radiodurans</i>), MntR (<i>Staphylococcus aureus</i>), MntR (<i>Escherichia coli</i>), and MntR (<i>Bacillus subtilis</i>). The sequences were aligned using the CLUSTAL W software. Residues shaded with black represent metal-binding sites that have been studied while residues shaded with grey represent predicted metal binding sites.</p

DR0865 binds to the promoter of MntABC in an ion-dependent manner.

<p>(A) and (B) DR0865 binding to p2523 and p2284 as the concentration of DR0865 increased. (C) Wild-type R1, <i>dr2539</i> null mutant (<b>Δ</b><i>dr2539</i>), and <i>dr0865</i> null mutant (<b>Δ</b><i>dr0865</i>) were cultured on TGY plates overlaid with filter discs saturated with 1 M solution MnCl₂. (D) The zone of inhibition was measured from edge of disc after three days. *, <i>P</i><0.05. Data represent the means±deviations of three independent experiments.</p

His98 plays an important role in DNA binding activity of DR2539.

<p>(A) 10 µl cell dilution was dripped on the TGY plate to which 6 mM of Mn(II) had been added. The cells were cultured for 3 days. (B) H98Y mutant and wild-type DR2539 proteins were incubated with p1709b at different concentrations of Mn(II). (C) Quantification of the fluorescence intensity of binding bands was performed using ImageJ. *, <i>P</i><0.05.</p

DR2539 binds to the MntH promoter DNA fragment in a Mn(II)- and Fe(II)-dependent manner.

<p>(A) Schematic of <i>dr1709</i> promoter (p1709a and p1709b) DNA sequence region. The inverted repeat region is shown by the inverted arrows. (B) DR2539 binding to p1709b with increasing quantities of DR2539 and 25 µM Mn(II). (C) and (D) EMSA analysis was performed using DR2539 and p1709b with increasing concentration of Mn(II) or Fe(II). RBS, ribosome binding site; Start, transcription start codon. p1709a and p1709b sequence regions are underlined by straight lines and dashed lines, respectively.</p

The interaction between DdrO and the promoter regions <i>in vivo</i>.

<p>QRT-PCR was performed using the immunoprecipitated DNA. DNA fragments cross-linked to DdrO were enriched by rabbit anti-DdrO antibody. Nonspecific normal antibody of rabbit in ChIP assay was applied as a blank control. <i>Dr0089</i> was used as an internal blank in QRT-PCR.</p

Comparison of DR1440 and P-type ATPase homologs.

(A) Multiple sequence alignments of DR1440 from D. radiodurans with P-type ATPase homologs. Sequences are from following bacteria: DR1440 from D. radiodurans, SsZntA from Shigella sonnei, BsZntA from Bacillus subtilis, LpCopA from Legionella pneumophila, EcCopA from Escherichia coli and TtCopA from Thermus thermophilus. The conserved metal binding sequence SPC is indicated by a dashed line box. Identical residues are shown as white letters with black background, and similar residues are shown as white letters with gray background. (B) The predicted structure of DR1440 obtained from homology modelling. The left panel shows the predicted structure of DR1440. Domain A is a Actuator domain, which contains a signature motif Thr-Gly-Glu (TGE) of P-type ATPases. Domain P contains conserved DKTG sequence related to the hydrolysis of ATP. Domain N performs ATP binding and phosphorylates the P-domain. The transmembrane (TM) domain consists of six membrane-spanning segments and harbours the metal ion binding sites. The right panel shows a close-up view of the TM helix containing the SPC metal binding site. CopA from L. pneumophila was used as a starting model.</p

Sensitivity of Δ<i>drb0067</i> strain subjected to H₂O₂.

<p>(A) and (B) Sensitivity of R1 and Δ<i>drb0067</i> to different concentration of H₂O₂. (C) and (D) Sensitivity of Δ<i>drb0067</i> to different concentration of H₂O₂ with the addition of 0.05 mM oligo(dG)50 or 2.5 mM dGMP. Data represent the means ± deviations of three independent experiments. R1, <i>D. radiodurans</i> wild type strain; Δ<i>drb0067</i>, the <i>drb0067</i> null mutant.</p