Live imaging of transcription sites using an elongating RNA polymerase II–specific probe

In eukaryotic nuclei, most genes are transcribed by RNA polymerase II (RNAP2), whose regulation is a key to understanding the genome and cell function. RNAP2 has a long heptapeptide repeat (Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7), and Ser2 is phosphorylated on an elongation form. To detect RNAP2 Ser2 phosphorylation (RNAP2 Ser2ph) in living cells, we developed a genetically encoded modification-specific intracellular antibody (mintbody) probe. The RNAP2 Ser2ph-mintbody exhibited numerous foci, possibly representing transcription “factories,” and foci were diminished during mitosis and in a Ser2 kinase inhibitor. An in vitro binding assay using phosphopeptides confirmed the mintbody’s specificity. RNAP2 Ser2ph-mintbody foci were colocalized with proteins associated with elongating RNAP2 compared with factors involved in the initiation. These results support the view that mintbody localization represents the sites of RNAP2 Ser2ph in living cells. RNAP2 Ser2ph-mintbody foci showed constrained diffusional motion like chromatin, but they were more mobile than DNA replication domains and p300-enriched foci, suggesting that the elongating RNAP2 complexes are separated from more confined chromatin domains.

UBE2S Activates NF-κB Signaling By Binding With IκBα and Promotes Metastasis of Lung Adenocarcinoma Cell

BackgroundNuclear factor (NF)-κB signaling in cancer cells was reported to be involved in tumorigenesis. Phosphorylation and degradation of inhibitor of NF-κBα (IκBα) is a canonical pathway of NF-κB signaling. Herein, we report non-canonical activation of NF-κB signaling without phosphorylation of IκB but by directly binding by ubiquitin-conjugating enzyme E2S (UBE2S) for degradation in adenocarcinoma cells.MethodsTCGA and the Human Atlas Protein Database were used to analyze the survival rate and expression of UBE2S. PC9, H460, H441 and A549 cells were used in this study. PC9 and H460 cells were used for further analysis because of different protein levels of UBE2S. Specific IKK inhibitors, PS1145 and SC514, were used to analyze the phosphorylation of IκBα. Western blotting experiment was used to analyze the protein levels PC9 and H460 cells. Wound-healing experiment was used to analyze the migrative ability of PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells were used to analyze the downstream proteins levels. Immunoprecipitation, immunofluorescent staining, a glutathione S transferase (GST) pull-down assay, and an in vitro binding assay were used to analyze the interaction of UBE2S and IκBα. Luciferase assay was used to analyze the activation of NF-κB signaling regulated by UBE2S. Zebrafish xenograft model was used analyzed the metastasis of PC9 cells regulated by UBE2S.Results UBE2S in lung adenocarcinoma patients was negatively related to the survival rate. The protein levels of UBE2S and IκBα were shown opposite change in PC9 and H460 cells. PC9 cells showed higher UBE2S expression and migrative ability than H460 cells. Phosphorylation of IκBα was not changed by treatment with IKK specific inhibitors, PS1145 and SC514, in PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells showed the protein levels of IκBα were regulated. Immunoprecipitation, immunofluorescent staining, a glutathione S transferase (GST) pull-down assay, and an in vitro binding assay showed the direct binding of UBE2S with IκBα. Protein levels of nuclear p65 and luciferase assay showed the NF-κB signaling was regulated UBE2S. EMT markers and migrative ability of cancer cells were also regulated by UBE2S. Zebrafish xenograft tumor model showed the reduction of migrative PC9 cells by knockdown of UBE2S.ConclusionHigher UBE2S expressed in lung adenocarcinomas could bind with IκBα for activation of NF-κB signaling to promote metastasis of cancer cells. UBE2S might be a potential therapeutic target for lung adenocarcinomas.

Repression of Wnt/β-catenin signaling by SOX9 and Mastermind-like transcriptional coactivator 2

Wnt/β-catenin signaling requires inhibition of a multiprotein destruction complex that targets β-catenin for proteasomal degradation. SOX9 is a potent antagonist of the Wnt pathway and has been proposed to act through direct binding to β-catenin or the β-catenin destruction complex. Here, we demonstrate that SOX9 promotes turnover of β-catenin in mammalian cell culture, but this occurs independently of the destruction complex and the proteasome. This activity requires SOX9’s ability to activate transcription. Transcriptome analysis revealed that SOX9 induces the expression of the Notch coactivator Mastermind-like transcriptional activator 2 (MAML2), which is required for SOX9-dependent Wnt/β-catenin antagonism. MAML2 promotes β-catenin turnover independently of Notch signaling, and MAML2 appears to associate directly with β-catenin in an in vitro binding assay. This work defines a previously unidentified pathway that promotes β-catenin degradation, acting in parallel to established mechanisms. SOX9 uses this pathway to restrict Wnt/β-catenin signaling.

Shape Effects of Peptide Amphiphile Micelles for Targeting Monocytes

Peptide amphiphile micelles (PAMs) are a nanoparticle platform that have gained popularity for their targeting versatility in a wide range of disease models. An important aspect of micelle design is considering the type of hydrophobic moiety used to synthesize the PAM, which can act as a contributing factor regarding their morphology and targeting capabilities. To delineate and compare the characteristics of spherical and cylindrical micelles, we incorporated the monocyte-targeting chemokine, monocyte chemoattractant protein-1 (MCP-1), into our micelles (MCP-1 PAMs). We report that both shapes of nanoparticles were biocompatible with monocytes and enhanced the secondary structure of the MCP-1 peptide, thereby improving the ability of the micelles to mimic the native MCP-1 protein structure. As a result, both shapes of MCP-1 PAMs effectively targeted monocytes in an in vitro binding assay with murine monocytes. Interestingly, cylindrical PAMs showed a greater ability to attract monocytes compared to spherical PAMs in a chemotaxis assay. However, the surface area, the multivalent display of peptides, and the zeta potential of PAMs may also influence their biomimetic properties. Herein, we introduce variations in the methods of PAM synthesis and discuss the differences in PAM characteristics that can impact the recruitment of monocytes, a process associated with disease and cancer progression.

Human ornithine decarboxylase paralogue (ODCp) is an antizyme inhibitor but not an arginine decarboxylase

ODC (ornithine decarboxylase), the rate-limiting enzyme in polyamine biosynthesis, is regulated by specific inhibitors, AZs (antizymes), which in turn are inhibited by AZI (AZ inhibitor). We originally identified and cloned the cDNA for a novel human ODC-like protein called ODCp (ODC paralogue). Since ODCp was devoid of ODC catalytic activity, we proposed that ODCp is a novel form of AZI. ODCp has subsequently been suggested to function either as mammalian ADC (arginine decarboxylase) or as AZI in mice. Here, we report that human ODCp is a novel AZI (AZIN2). By using yeast two-hybrid screening and in vitro binding assay, we show that ODCp binds AZ1–3. Measurements of the ODC activity and ODC degradation assay reveal that ODCp inhibits AZ1 function as efficiently as AZI both in vitro and in vivo. We further demonstrate that the degradation of ODCp is ubiquitin-dependent and AZ1-independent similar to the degradation of AZI. We also show that human ODCp has no intrinsic ADC activity.