Description: Most previously reported methods for purifying DNA-origami nanostructures rely on agarose-gel electrophoresis (AGE) for separation. Although AGE is routinely used to yield 0.1–1 µg purified DNA nanostructures, obtaining 〉100 µg of purified DNA-origami structure through AGE is typically laborious because of the post-electrophoresis extraction, desalting and concentration steps. Here, we present a readily scalable purification approach utilizing rate-zonal centrifugation, which provides comparable separation resolution as AGE. The DNA nanostructures remain in aqueous solution throughout the purification process. Therefore, the desired products are easily recovered with consistently high yield (40–80%) and without contaminants such as residual agarose gel or DNA intercalating dyes. Seven distinct three-dimensional DNA-origami constructs were purified at the scale of 0.1–100 µg (final yield) per centrifuge tube, showing the versatility of this method. Given the commercially available equipment for gradient mixing and fraction collection, this method should be amenable to automation and further scale up for preparation of larger amounts (e.g. milligram quantities) of DNA nanostructures.

Description: High-mobility group box 1 protein (HMGB1), which mainly exists in the nucleus, has recently been shown to function as a sentinel molecule for viral nucleic acid sensing and an autophagy regulator in the cytoplasm. In this study, we studied the chaperone-like activity of HMGB1 and found that HMGB1 inhibited the chemically induced aggregation of insulin and lysozyme, as well as the heat-induced aggregation of citrate synthase. HMGB1 also restored the heat-induced suppression of cytoplasmic luciferase activity as a reporter protein in hamster lung fibroblast O23 cells with expression of HMGB1. Next, we demonstrated that HMGB1 inhibited the formation of aggregates and toxicity caused by expanded polyglutamine (polyQ), one of the main causes of Huntington disease. HMGB1 directly interacted with polyQ on immunofluorescence and coimmunoprecipitation assay, whereas the overexpression of HMGB1 or exogenous administration of recombinant HMGB1 protein remarkably reduced polyQ aggregates in SHSY5Y cells and hmgb1 –/– mouse embryonic fibroblasts upon filter trap and immunofluorescence assay. Finally, overexpressed HMGB1 proteins in mouse embryonic primary striatal neurons also bound to polyQ and decreased the formation of polyQ aggregates. To this end, we have demonstrated that HMGB1 exhibits chaperone-like activity and a possible therapeutic candidate in polyQ disease.

Description: Smad3, a major intracellular mediator of TGFβ signaling, functions as both a positive and negative regulator in carcinogenesis. In response to TGFβ, the TGFβ receptor phosphorylates serine residues at the Smad3 C-tail. Cancer cells often contain high levels of the MAPK and CDK activities, which can lead to the Smad3 linker region becoming highly phosphorylated. Here, we report, for the first time, that mutation of the Smad3 linker phosphorylation sites markedly inhibited primary tumor growth, but significantly increased lung metastasis of breast cancer cell lines. In contrast, mutation of the Smad3 C-tail phosphorylation sites had the opposite effect. We show that mutation of the Smad3 linker phosphorylation sites greatly intensifies all TGFβ-induced responses, including growth arrest, apoptosis, reduction in the size of putative cancer stem cell population, epithelial–mesenchymal transition, and invasive activity. Moreover, all TGFβ responses were completely lost on mutation of the Smad3 C-tail phosphorylation sites. Our results demonstrate a critical role of the counterbalance between the Smad3 C-tail and linker phosphorylation in tumorigenesis and metastasis. Our findings have important implications for therapeutic intervention of breast cancer. Cancer Res; 74(21); 6139–49. ©2014 AACR.

Description: Canonical transient receptor potential (TRPC) channels are Ca2+-permeable nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). The TRPC4 channel is expressed in a punctate distribution in the membrane. To identify the regulating region of the channel trafficking to the membrane, we generated deletion mutants of the TRPC4 channel. We determined that when either region that was downstream of the 20 amino acids of the N terminus or the 700–730 amino acids was deleted, the mutants were retained in the endoplasmic reticulum. By coexpression of the wild-type TRPC4 with deletion mutants, we found that the 23–29 amino acids of the N terminus regulate a membrane trafficking. Additionally, by the fluorescence resonance energy transfer (FRET) method, we found that the regions downstream of the 99 amino acid region of the N terminus and upstream of the 730 amino acid region in the C terminus produce assembly of the TRPC4 tetramers. We inferred the candidate proteins that regulate or interact with the 23–29 domain of TRPC4.

Description: Overexpression of BCRP/ABCG2, a xenobiotic efflux transporter, is associated with anticancer drug resistance in tumors. Proto-oncogene c-MET induces cancer cell proliferation, motility, and survival, and its aberrant activation was found to be a prognostic factor in advanced ovarian cancers. In the present study, we investigated the potential crossresistance of doxorubicin-resistant ovarian cancer cells to the pheophorbide a (Pba)–based photodynamic therapy (PDT), and suggest c-MET and BCRP/ABCG2 overexpression as an underlying molecular mechanism. The doxorubicin-resistant A2780 cell line (A2780DR), which was established by incubating A2780 with stepwise increasing concentrations of doxorubicin, showed low levels of cellular Pba accumulation and reactive oxygen species generation, and was more resistant to PDT cytotoxicity than A2780. In a microarray analysis, BCRP/ABCG2 was found to be the only drug transporter whose expression was upregulated in A2780DR; this increase was confirmed by Western blot and immunocytochemical analyses. As functional evidence, the treatment with a BCRP/ABCG2-specific inhibitor reversed A2780DR resistance to both doxorubicin and PDT. We identified that c-MET increase is related to BCRP/ABCG2 activation. The c-MET downstream phosphoinositide 3-kinase (PI3K)/AKT signaling was activated in A2780DR and the inhibition of PI3K/AKT or c-MET repressed resistance to doxorubicin and PDT. Finally, we showed that the pharmacological and genetic inhibition of c-MET diminished levels of BCRP/ABCG2 in A2780DR. Moreover, c-MET inhibition could repress BCRP/ABCG2 expression in breast carcinoma MDA-MB-231 and colon carcinoma HT29, resulting in sensitization to doxorubicin. Collectively, our results provide a novel link of c-MET overexpression to BCRP/ABCG2 activation, suggesting that this mechanism leads to crossresistance to both chemotherapy and PDT.

Description: High mobility group nucleosomal binding domain 2 (HMGN2) is a small and unique non-histone protein that has many functions in a variety of cellular processes, including regulation of chromatin structure, transcription, and DNA repair. In addition, it may have other roles in antimicrobial activity, cell homing, and regulating cytokine release. Although the biochemical properties of HMGN2 protein are regulated by acetylation and phosphorylation, it is not yet known whether HMGN2 activity can also be regulated by SUMOylation. In this study, we demonstrated for the first time that HMGN2 is modified by covalent attachment of small ubiquitin-related modifier 1 (SUMO1) by pro-inflammatory signal and identified the major SUMOylated lysine residues that localize to the HMGN2 nucleosome-binding domain at Lys-17 and Lys-35. SENP1 can deSUMOylate SUMOylated HMGN2, and PIAS1 is the E3 ligase responsible for SUMOylation of HMGN2. Finally, using SUMO1-conjugated HMGN2 purified from a basal SUMOylation system in Escherichia coli, we demonstrated that SUMOylated HMGN2 has decreased the binding affinity to nucleosome core particles in comparison to unSUMOylated HMGN2. These observations potentially provide new perspectives for understanding the functions of HMGN2 in inflammatory reaction.

Description: TPRC channels are Ca 2+ -permeable, nonselective cation channels that are activated by a wide variety of stimuli, including G protein-coupled receptors (GPCRs). TRPC4 is commonly assumed to be activated by G q /phospholipase C-coupled receptors. However, the other molecular mechanisms by which Gα proteins regulate TRPC4 remain unclear. Here, we found that Gα i2 regulates TRPC4 activation by direct binding. To investigate this mechanism, we used whole patch clamp and fluorescence resonance energy transfer (FRET). We tagged an isoform of mTRPC4 and G protein with CFP and YFP, respectively, and transiently transfected cells with the FRET pair. The FRET efficiency between TRPC4β-CFP and the constitutively active mutant form of Gα i2 was nearly 15% and was greater than that observed with wild-type Gα i2 (nearly 5%). Gβ and the TRPC4 channel showed a FRET efficiency lower than 6%. In HEK293 cells transfected with the M2 muscarinic receptor, the application of carbachol increased the FRET efficiency between TRPC4β-CFP and Gα i2 (WT)-YFP from 4.7 ± 0.4% ( n = 7) to 12.6 ± 1.4% ( n = 7). We also found that the TRPC4 channel directly interacts with Gα i2 , but not with Gα q , when the channel is open. We analyzed the calcium levels in HEK293 cells expressing the channels and Gα i2 or Gα q using the calcium indicator YC6.1 (Yellow Cameleon 6.1). In response to the muscarinic agonist carbachol, M2-, Gα i2 -, and TRPC4-expressing cells showed a prolonged Ca 2+ influx compared with cells expressing only M2. Together, these data suggest that Gα i2 activates the TRPC4 channel by direct binding, which then induces Ca 2+ entry.

Description: Context Previous studies using autologous bone marrow mononuclear cells (BMCs) in patients with ischemic cardiomyopathy have demonstrated safety and suggested efficacy. Objective To determine if administration of BMCs through transendocardial injections improves myocardial perfusion, reduces left ventricular end-systolic volume (LVESV), or enhances maximal oxygen consumption in patients with coronary artery disease or LV dysfunction, and limiting heart failure or angina. Design, Setting, and Patients A phase 2 randomized double-blind, placebo-controlled trial of symptomatic patients (New York Heart Association classification II-III or Canadian Cardiovascular Society classification II-IV) with a left ventricular ejection fraction of 45% or less, a perfusion defect by single-photon emission tomography (SPECT), and coronary artery disease not amenable to revascularization who were receiving maximal medical therapy at 5 National Heart, Lung, and Blood Institute–sponsored Cardiovascular Cell Therapy Research Network (CCTRN) sites between April 29, 2009, and April 18, 2011. Intervention Bone marrow aspiration (isolation of BMCs using a standardized automated system performed locally) and transendocardial injection of 100 million BMCs or placebo (ratio of 2 for BMC group to 1 for placebo group). Main Outcome Measures Co-primary end points assessed at 6 months: changes in LVESV assessed by echocardiography, maximal oxygen consumption, and reversibility on SPECT. Phenotypic and functional analyses of the cell product were performed by the CCTRN biorepository core laboratory. Results Of 153 patients who provided consent, a total of 92 (82 men; average age: 63 years) were randomized (n = 61 in BMC group and n = 31 in placebo group). Changes in LVESV index (–0.9 mL/m 2 [95% CI, –6.1 to 4.3]; P  = .73), maximal oxygen consumption (1.0 [95% CI, –0.42 to 2.34]; P  = .17), and reversible defect (–1.2 [95% CI, –12.50 to 10.12]; P  = .84) were not statistically significant. There were no differences found in any of the secondary outcomes, including percent myocardial defect, total defect size, fixed defect size, regional wall motion, and clinical improvement. Conclusion Among patients with chronic ischemic heart failure, transendocardial injection of autologous BMCs compared with placebo did not improve LVESV, maximal oxygen consumption, or reversibility on SPECT. Trial Registration clinicaltrials.gov Identifier: NCT00824005