Evidence for DNA Cleavage Caused Directly by a transfer RNA-Targeting Toxin

<div><p>The killer yeast species <i>Pichia</i><i>acaciae</i> produces a heteromeric killer protein, PaT, that causes DNA damage and arrests the cell cycle of sensitive <i>Saccharomyces cerevisiae</i> in the S phase. However, the mechanism by which DNA damage occurs remains elusive. A previous study has indicated that Orf2p, a subunit of PaT, specifically cleaves an anticodon loop of an <i>S. cerevisiae</i> transfer RNA (tRNA^Gln_mcm5s2UUG). This finding raised a question about whether the DNA damage is a result of the tRNA cleavage or whether Orf2p directly associates with and cleaves the genomic DNA of sensitive yeast cells. We showed that Orf2p cleaves genomic DNA in addition to cleaving tRNA <i>in vitro</i>. This DNA cleavage requires the same Orf2p residue as that needed for tRNA cleavage, His299. The expression of Orf2p, in which His299 was substituted to alanine, abolished the cell cycle arrest of the host cell. Moreover, the translation impairment induced by tRNA cleavage enabled Orf2p to enter the nucleus, thereby inducing histone phosphorylation.</p> </div

Translation impairment allows nuclear translocation of Orf2p.

<p>(A) Wild-type (WT) Orf2p and Orf2p-H299A, which are Flag-tagged at the C-terminus, were expressed in CG379 cells as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075512#pone-0075512-g002" target="_blank">Figure 2B</a> and detected with western blotting at each time point. Wild-type Orf2p was not detected even after 4 hours of expression. (B) Expression of Orf2p-H299A was induced for 5 hours in cells as shown in (A) followed by 1 hour of cycloheximide treatment as indicated to mimic the translation impairment caused by tRNA cleavage. After nucleus fractionation, Flag-tagged Orf2p-H299A was detected with western blotting. Pgk1p and histone H4 were detected as markers in the cytosol and nucleus, respectively. WCE indicates unfractionated lysate. Band intensities of Orf2p in the nucleus were remarkable with cycloheximide treatment, indicating that the translocation of Orf2p into the nucleus is translation-impairment dependent.</p

Orf2p cleaves DNA via His299 <i>in vitro</i>.

<p>(A) Wild-type Orf2p, Orf2p-H299A, and γ-subunit were incubated with total RNA prepared from <i>S. cerevisiae</i>. Then, northern hybridization was carried out for the detection of tRNAs, as indicated. Modification of the first letter of tRNA^Glu_mcm5s2UUC and tRNA^Gln_mcm5s2UUG was omitted for simplicity of labeling. Purified Orf2p retained substrate specificity, and Orf2p-H299A lost tRNA cleavage activity. (B) Wild-type (WT) Orf2p, Orf2p-H299A, and γ-subunit were incubated with <i>Saccharomyces cerevisiae</i> genomic DNA and subjected to agarose gel electrophoresis. (C) Linearized pGMH10 was used as a substrate instead of genomic DNA. After incubation with Orf2p, DNA was extracted with phenol/chloroform treatment and electrophoresed (middle panel in [B] and right panel in [C]). Linearized λ phage DNA digested with <i>Sty</i>I was loaded as a size marker and is indicated as M. Both Orf2p and Orf2p-H299A slowed the electrophoretic mobility of substrate DNA. Moreover, the band became faint after incubation with wild-type Orf2p, but not with its mutant, indicating that DNA cleavage activity of Orf2p depends on His299.</p

Orf2p induces histone phosphorylation.

<p>(A) Transfer RNA (tRNA)-targeting toxins were chromosomally expressed in CG379 cells, and the status of histone H2A phosphorylation was compared using western blotting. Pronounced histone phosphorylation was observed in cells expressing Orf2p. MagicMark XP (Invitrogen) was used as a molecular weight marker. (B) TM142 cells were challenged with zymocin, and phosphorylation of histone was analyzed as in (A). Histone was phosphorylated in cells challenged with zymocin for 2 hours. Methylmethane sulfonate (MMS) was used to induce DNA damage as a control.</p

His299 of Orf2p is responsible for tRNA cleavage and DNA damage induction.

<p>(A) Four Orf2p mutants, in which each of the His residues was replaced with Ala, were expressed by a high-copy plasmid in TM142 cells, and the viability of these transformants was compared. Orf2p-H299A completely abolished growth impairment. (B) Total tRNA was prepared from CG379 cells chromosomally expressing wild-type (WT) Orf2p or Orf2p-H299A at each interval, and tRNA^Gln_mcm5s2UUG cleavage was observed using northern hybridization. Modification of the first letter of tRNA^Gln_mcm5s2UUG was omitted for simplicity of labeling. Orf2p-H299A did not cleave tRNA^Gln_mcm5s2UUG, showing that His299 is indispensable for tRNA cleavage. (C) Wild-type or mutant Orf2p were expressed for 12 hours as shown in (B), and then cells were collected and status of cell cycle was analyzed with fluorescence-activated cell sorting. The cell number is shown on the vertical axis, and the horizontal axis indicates the fluorescence intensity of nuclei stained with propidium iodide. Cell cycle progression of cells in which Orf2p-H299A was expressed was not impaired. (D) Phosphorylation of histone in cells expressing wild-type and mutant Orf2p was determined with western blotting at the indicated time points, as shown in (B). A DNA damage response was not induced in cells expressing Orf2p-H299A, as indicated by a lack of phosphorylation.</p

AJICAP-M: Traceless Affinity Peptide Mediated Conjugation Technology for Site-Selective Antibody–Drug Conjugate Synthesis

A traceless site-selective conjugation method, “AJICAP-M”, was developed for native antibodies at sites using Fc-affinity peptides, focusing on Lys248 or Lys288. It produces antibody–drug conjugates (ADCs) with consistent drug-to-antibody ratios, enhanced stability, and simplified manufacturing. Comparative in vivo assessment demonstrated AJICAP-M’s superior stability over traditional ADCs. This technology has been successfully applied to continuous-flow manufacturing, marking the first achievement in site-selective ADC production. This manuscript outlines AJICAP-M’s methodology and its effectiveness in ADC production

Interaction Analysis of FABP4 Inhibitors by X‑ray Crystallography and Fragment Molecular Orbital Analysis

X-ray crystal structural determination of FABP4 in complex with four inhibitors revealed the complex binding modes, and the resulting observations led to improvement of the inhibitory potency of FABP4 inhibitors. However, the detailed structure–activity relationship (SAR) could not be explained from these structural observations. For a more detailed understanding of the interactions between FABP4 and inhibitors, fragment molecular orbital analyses were performed. These analyses revealed that the total interfragment interaction energies of FABP4 and each inhibitor correlated with the ranking of the <i>K</i>_i value for the four inhibitors. Furthermore, interactions between each inhibitor and amino acid residues in FABP4 were identified. The oxygen atom of Lys58 in FABP4 was found to be very important for strong interactions with FABP4. These results might provide useful information for the development of novel potent FABP4 inhibitors