Effect of K63 polyubiquitination on the quality control for stalled translation.

<p>(A) Structure of Ubiquitin protein and location of the lysine residues for polyubiquitination [id: 1UBQ]. K63 (red) and other lysine residues (pink) are shown in sticks. The structural model was generated using MolFeat version 3.5 (Fiatlux, Tokyo, Japan). (B) Schematic illustration for the inhibition of polyubiquitination by arginine mutants. Arginine mutants were expressed from expression plasmids in yeast cells harboring endogenous ubiquitin genes on its genome. An incorporation of an arginine mutant into the (poly-) ubiquitin chain terminates further elongation. (C) Cellular ubiquitination levels of wild-type and ubiquitin mutants with a single arginine substitution (K6R, K11R, K27R, K29R, K33R, K48R, K63R). Ubiquitin genes were fused with His-tag and expressed from plasmids in the wild-type strain (BY4727). Cellular ubiquitination levels were detected by antibody for His-tag. PGK antibody was used as loading control. (D) Dual luciferase assay for the effects of arginine mutants of ubiquitin protein on the Rluc-CGA x12-luc2 reporter. Plasmids that included ubiquitin arginine mutants were introduced into a wild-type strain (HRKW-2). Ubi-WT indicates wild-type ubiquitin. Average luc2/Rluc ratios and standard deviations were determined from three independent measurements.</p

Inhibiting K63 Polyubiquitination Abolishes No-Go Type Stalled Translation Surveillance in <i>Saccharomyces cerevisiae</i>

<div><p>Incidental ribosome stalling during translation elongation is an aberrant phenomenon during protein synthesis and is subjected to quality control by surveillance systems, in which mRNA and a nascent protein are rapidly degraded. Their detailed molecular mechanisms as well as responsible factors for these processes are beginning to be understood. However, the initial processes for detecting stalled translation that result in degradation remain to be determined. Among the factors identified to date, two E3 ubiquitin ligases have been reported to function in distinct manners. Because ubiquitination is one of the most versatile of cellular signals, these distinct functions of E3 ligases suggested diverse ubiquitination pathways during surveillance for stalled translation. In this study, we report experimental evidences for a unique role of non-proteasomal K63 polyubiquitination during quality control for stalled translation. Inhibiting K63 polyubiquitination by expressing a K63R ubiquitin mutation in <i>Saccharomyces cerevisiae</i> cells markedly abolished the quality control responses for stalled translation. More detailed analyses indicated that the effects of K63R mutants were independent of the proteasome and that K63 polyubiquitination is dependent on Hel2, one of the E3 ligases. Moreover, a K63R ubiquitin mutant barely inhibited the quality control pathway for nonstop translation, indicating distinct mechanisms for these highly related quality control pathways. Our results suggest that non-proteasomal K63 polyubiquitination is included in the initial surveillance process of stalled translation and presumably triggers protein degradation steps upon translational stall. These findings provide crucial information regarding the detailed molecular mechanisms for the initial steps involved in quality control systems and their classification.</p></div

Comparisons of the effects of deleting Hel2 and Ltn1.

<p>(A) Western blot analysis for Rluc-HIS3 reporters from the wild-type (WT), <i>hel2</i><b><i>∆</i></b>, and <i>ltn1</i><b><i>∆</i></b> strains (BY4727, SKY61, S18-E01). A schematic image of reporter fusion genes, Rluc-HIS3, either with or without 12 CGA codon repeats, is illustrated above. The expression of fusion protein was detected using a Rluc antibody. PGK1 was used as a loading control. Arrowhead 1 indicates the Rluc protein alone, arrowhead 2 indicates the Rluc-blank-HIS3 protein, and arrowhead 3 indicates the Rluc-CGA x12-HIS3 protein. (B) Dual luciferase assay for Rluc-CGA x12-luc2 reporter in the wild-type (WT), <i>hel2</i><b><i>∆</i></b>, and <i>ltn1</i><b><i>∆</i></b> strains (HRKW-2, SKY113, HRKW-6). A schematic image of reporter fusion genes, Rluc-luc2, either with or without 12 CGA codon repeats, is illustrated above. Bars indicate luc2/Rluc ratios. Percentages were standardized using the results of a dual luciferase assay for Rluc-blank-luc2 in the wild-type strain (HRKW-1). The inset indicates the results with the <i>ltn1</i><b><i>∆</i></b> strain with an appropriately adjusted range. Average luc2/Rluc ratios and standard deviations were determined from three independent measurements.</p

Effects of proteasome inhibitors, MG132 and PS341, on stalled translation and K63R expression.

<p>(A) Western blot analysis for effect of MG132. Vectors with Rluc-HIS3 reporters were introduced into a wild-type strain (BY4727). Yeast transformants were incubated for 5 h with either DMSO (e.g., 0 μM MG132) or 75 μM MG132. Arrowhead 1 indicates the Rluc protein only and arrowhead 2 indicates the Rluc-blank-HIS3 protein. PGK1 was used as a loading control. (B) Dual luciferase assay for the effect of MG132 on wild-type strain with Rluc-CGA x12-luc2 reporter (HRKW-2). MG132 was used as in (A). (C) The empty or K63R ubiquitin mutation plasmids were introduced into the <i>pdr5</i><b><i>∆</i></b> yeast strain harboring Rluc-luc2 reporter (SKY142). The transformants were incubated in the presence of MG132 or PS341 at indicated concentrations for 2 h. Fold differences between the percentages of luc2/Rluc ratios between the transformants with the absence (-K63R) or presence (+K63R) of K63R expression are indicated over the top of the bar graphs.</p

Effects of K0 and K63only ubiquitin mutants on stalled translation.

<p>(A) Cellular ubiquitination levels of K0 and K63only ubiquitin mutants. Ubiquitinated proteins were detected as above. Amount of the protein sample from a cell expressing His-tagged wild-type ubiquitin are decreased to 1/16 and 1/4 dilution. (B) Dual luciferase assay for the of K0 and K63only ubiquitin mutants on the Rluc-luc2 reporter. Plasmids bearing K0 or K63only ubiquitin mutants were introduced into a wild-type strain harboring Rluc-luc2 reporter strain (HRKW-2). (single) indicates the transformant with ubiquitin expression from single copy plasmids, and (multi) indicates that from multiple copy plasmids. Average luc2/Rluc ratios and standard deviations were determined from three independent measurements. (C) Genetic colony growth test for the effect of K0 and K63only ubiquitin mutants on the Rluc-HIS3 reporter (SKY24). Types of ubiquitin vectors are indicated on the left. Transformant colonies were streaked on the SC-Leucine (Leu) (middle), and SC-Leucine, Histidine (Leu, His) (right) plates, and colony growth was monitored for 4 days at 30°C.</p

Temporal regulation of K63 polyubiquitination and Hel2 with other factors for stalled translation.

<p>(A) The luc2/Rluc ratios for Rluc-luc2 reporter in the wild-type (WT) and single knockout strains (as indicated) in the presence (+K63R) or absence (-K63R) of K63R expression. The inset shows the results for the <i>rqc1</i><b><i>∆</i></b> and <i>ltn1</i><b><i>∆</i></b> strains. The average luc2/Rluc ratios and their standard deviations were obtained for three independent measurements. (B) The luc2/Rluc ratios for Rluc-luc2 reporter in the strains with double knockout of Hel2 and other factors involved in stalled translation. All strains are <i>hel2</i><b><i>∆</i></b>. Thus, as examples, WT in this figure indicates a <i>hel2</i><b><i>∆</i></b> single knockout strain, and <i>asc1</i><b><i>∆</i></b> indicates the <i>hel2</i><b><i>∆</i></b><i>asc1</i><b><i>∆</i></b> strain. The average luc2/Rluc ratios and their standard deviations were obtained for three independent measurements. *<i>p</i> < 0.05, **<i>p</i> < 0.01.</p

SourceCode_KI_etal_2016

Source code for the analysis in "The evolution of cooperation by negotiation in a noisy world"

Additional file 1: Figure S1. of PRKCQ promotes oncogenic growth and anoikis resistance of a subset of triple-negative breast cancer cells

PRKCQ downregulation in MDA-231-LucD3H2LN cells results in G2-M arrest. Triple-negative MDA-231-LucD3H2LN cells were infected with PRKCQ shRNA lentiviral supernatant for 48 hours. Cells were fixed and stained with PI, and cell-cycle profile analysis was performed using flow cytometry. Results from four independent experiments were averaged. Figure S2 PRKCQ shRNA has no effect on growth of MDA231 cells that do not express detectable PKCθ protein. MDA231 cells expressing empty vector (EV) or PRKCQ shRNA (54) were grown in 3-D cultures for 7 days. (PDF 573 kb

Dual-sgRNA CRISPRa System for Enhanced MK‑7 Production and <i>Salmonella</i> Infection Mitigation in <i>Bacillus subtilis</i> natto Applied to Caco‑2 Cells

This study optimized the menaquinone-7 (MK-7) synthetic pathways in Bacillus subtilis (B. subtilis) natto NB205, a strain that originated from natto, to enhance its MK-7 production. Utilizing mutation breeding, we developed NBMK308, a mutant strain that demonstrated a significant 117.23% increase in MK-7 production. A comprehensive transcriptome analysis identified two key genes, ispA and ispE, as being critical in MK-7 synthesis. The dual-sgRNA CRISPRa system was utilized to achieve precise regulation of ispA and ispE in the newly engineered strain, A3E3. This strategic modulation resulted in a significant enhancement of MK-7 production, achieving increases of 20.02% and 201.41% compared to traditional overexpression systems and the original strain NB205, respectively. Furthermore, the fermentation supernatant from A3E3 notably inhibited Salmonella invasion in Caco-2 cells, showcasing its potential for combating such infections. The safety of the dual-sgRNA CRISPRa system was confirmed through cell assays. The utilization of the dual-sgRNA CRISPRa system in this study was crucial for the precise regulation of key genes in MK-7 synthesis, leading to a remarkable increase in production and demonstrating additional therapeutic potential in inhibiting pathogenic infections. This approach effectively combined the advantages of microbial fermentation and biotechnology, addressing health and nutritional challenges

Sp1 plays an important role in nicotine-mediated LDLR expression.

<p>(A) Ca9-22 cells were pre-treated with or without mithramycin for 1 h. After washes, the cells were stimulated with 100 μM of nicotine for 3 h and luciferase activity was measured. (B) Ca9-22 cells were transfected with various concentrations of siRNA against Sp1 or control siRNA (con) for 3 h. After transfection, the cells were stimulated with or without 100 μM of nicotine for 3 h. The expression level of LDLR mRNA was examined by real-time PCR. (C) The silencing effect of siRNA transfection on the expression of Sp1 was assessed by real-time PCR. Sp1 expression level of control siRNA-transfected Ca9-22 cell was set as 1. The data are presented as mean ± SD of at least 3 separate experiments. *p < 0.05.</p