Browsing by Subject "Blotting, Western"

Now showing 1 - 5 of 5
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    Development of AlphaLISA high throughput technique to screen for small molecule inhibitors targeting protein arginine methyltransferases
    (Royal Society of Chemistry, 2017-11-21) Prabhu, Lakshmi; Chen, Lan; Wei, Han; Demir, Özlem; Safa, Ahmad; Zeng, Lifan; Amaro, Rommie E.; O’Neil, Bert H.; Zhang, Zhongyin; Lu, Tao; Pharmacology and Toxicology, School of Medicine
    The protein arginine methyltransferase (PRMT) family of enzymes comprises nine family members in mammals. They catalyze arginine methylation, either monomethylation or symmetric/asymmetric dimethylation of histone and non-histone proteins. PRMT methylation of its substrate proteins modulates cellular processes such as signal transduction, transcription, and mRNA splicing. Recent studies have linked overexpression of PRMT5, a member of the PRMT superfamily, to oncogenesis, making it a potential target for cancer therapy. In this study, we developed a highly sensitive (Z' score = 0.7) robotic high throughput screening (HTS) platform to discover small molecule inhibitors of PRMT5 by adapting the AlphaLISA™ technology. Using biotinylated histone H4 as a substrate, and S-adenosyl-l-methionine as a methyl donor, PRMT5 symmetrically dimethylated H4 at arginine (R) 3. Highly specific acceptor beads for symmetrically dimethylated H4R3 and streptavidin-coated donor beads bound the substrate, emitting a signal that is proportional to the methyltransferase activity. Using this powerful approach, we identified specific PRMT5 inhibitors P1608K04 and P1618J22, and further validated their efficacy and specificity for inhibiting PRMT5. Importantly, these two compounds exhibited much more potent efficacy than the commercial PRMT5 inhibitor EPZ015666 in both pancreatic and colorectal cancer cells. Overall, our work highlights a novel, powerful, and sensitive approach to identify specific PRMT5 inhibitors. The general principle of this HTS screening method can not only be applied to PRMT5 and the PRMT superfamily, but may also be extended to other epigenetic targets. This approach allows us to identify compounds that inhibit the activity of their respective targets, and screening hits like P1608K04 and P1618J22 may serve as the basis for novel drug development to treat cancer and/or other diseases.
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    Glucocorticoid hormone-induced chromatin remodeling enhances human hematopoietic stem cell homing and engraftment
    (Nature Publishing Group, 2017-04) Guo, Bin; Huang, Xinxin; Cooper, Scott; Broxmeyer, Hal E.; Microbiology and Immunology, School of Medicine
    Efficient hematopoietic stem cell (HSC) homing is important for hematopoietic cell transplantation (HCT), especially when HSC numbers are limited, as in the use of cord blood (CB). In a screen of small-molecule compounds, we identified glucocorticoid (GC) hormone signaling as an activator of CXCR4 expression in human CB HSCs and hematopoietic progenitor cells (HPCs). Short-term GC pretreatment of human CB HSCs and HPCs promoted SDF-1-CXCR4-axis-mediated chemotaxis, homing, and long-term engraftment when these cells were transplanted into primary- and secondary-recipient NSG mice. Mechanistically, activated glucocorticoid receptor binds directly to a glucocorticoid response element in the CXCR4 promoter and recruits the SRC-1-p300 complex to promote H4K5 and H4K16 histone acetylation, facilitating transcription of CXCR4. These results suggest a new and readily available means to enhance the clinical efficacy of CB HCT.
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    Nuclear Factor Erythroid 2-Related Factor 2 Deficiency Results in Amplification of the Liver Fat-Lowering Effect of Estrogen
    (American Society for Pharmacology and Experimental Therapeutics, 2016-07) Rui, Wenjuan; Zou, Yuhong; Lee, Joonyong; Nambiar, Shashank Manohar; Lin, Jingmei; Zhang, Linjie; Yang, Yan; Dai, Guoli; Biology, School of Science
    Transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates multiple biologic processes, including hepatic lipid metabolism. Estrogen exerts actions affecting energy homeostasis, including a liver fat-lowering effect. Increasing evidence indicates the crosstalk between these two molecules. The aim of this study was to evaluate whether Nrf2 modulates estrogen signaling in hepatic lipid metabolism. Nonalcoholic fatty liver disease (NAFLD) was induced in wild-type and Nrf2-null mice fed a high-fat diet and the liver fat-lowering effect of exogenous estrogen was subsequently assessed. We found that exogenous estrogen eliminated 49% and 90% of hepatic triglycerides in wild-type and Nrf2-null mice with NAFLD, respectively. This observation demonstrates that Nrf2 signaling is antagonistic to estrogen signaling in hepatic fat metabolism; thus, Nrf2 absence results in striking amplification of the liver fat-lowering effect of estrogen. In addition, we found the association of trefoil factor 3 and fatty acid binding protein 5 with the liver fat-lowering effect of estrogen. In summary, we identified Nrf2 as a novel and potent inhibitor of estrogen signaling in hepatic lipid metabolism. Our finding may provide a potential strategy to treat NAFLD by dually targeting Nrf2 and estrogen signaling.
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    Ras-Mek-Erk Signaling Regulates Nf1 Heterozygous Neointima Formation
    (Elsevier B.V., 2014-01) Stansfield, Brian K.; Bessler, Waylan K.; Mali, Raghuveer; Mund, Julie A.; Downing, Brandon D.; Kapur, Reuben; Ingram, David A. Jr; Department of Pediatrics, IU School of Medicine
    Neurofibromatosis type 1 (NF1) results from mutations in the NF1 tumor-suppressor gene, which encodes neurofibromin, a negative regulator of diverse Ras signaling cascades. Arterial stenosis is a nonneoplastic manifestation of NF1 that predisposes some patients to debilitating morbidity and sudden death. Recent murine studies demonstrate that Nf1 heterozygosity (Nf1+/−) in monocytes/macrophages significantly enhances intimal proliferation after arterial injury. However, the downstream Ras effector pathway responsible for this phenotype is unknown. Based on in vitro assays demonstrating enhanced extracellular signal-related kinase (Erk) signaling in Nf1+/− macrophages and vascular smooth muscle cells and in vivo evidence of Erk amplification without alteration of phosphatidylinositol 3-kinase signaling in Nf1+/− neointimas, we tested the hypothesis that Ras-Erk signaling regulates intimal proliferation in a murine model of NF1 arterial stenosis. By using a well-established in vivo model of inflammatory cell migration and standard cell culture, neurofibromin-deficient macrophages demonstrate enhanced sensitivity to growth factor stimulation in vivo and in vitro, which is significantly diminished in the presence of PD0325901, a specific inhibitor of Ras-Erk signaling in phase 2 clinical trials for cancer. After carotid artery injury, Nf1+/− mice demonstrated increased intimal proliferation compared with wild-type mice. Daily administration of PD0325901 significantly reduced Nf1+/− neointima formation to levels of wild-type mice. These studies identify the Ras-Erk pathway in neurofibromin-deficient macrophages as the aberrant pathway responsible for enhanced neointima formation.
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    Titer estimation for quality control (TEQC) method: A practical approach for optimal production of protein complexes using the baculovirus expression vector system
    (Public Library of Science, 2018-04-03) Imasaki, Tsuyoshi; Wenzel, Sabine; Yamada, Kentaro; Bryant, Megan L.; Takagi, Yuichiro; Biochemistry and Molecular Biology, School of Medicine
    The baculovirus expression vector system (BEVS) is becoming the method of choice for expression of many eukaryotic proteins and protein complexes for biochemical, structural and pharmaceutical studies. Significant technological advancement has made generation of recombinant baculoviruses easy, efficient and user-friendly. However, there is a tremendous variability in the amount of proteins made using the BEVS, including different batches of virus made to express the same proteins. Yet, what influences the overall production of proteins or protein complexes remains largely unclear. Many downstream applications, particularly protein structure determination, require purification of large quantities of proteins in a repetitive manner, calling for a reliable experimental set-up to obtain proteins or protein complexes of interest consistently. During our investigation of optimizing the expression of the Mediator Head module, we discovered that the 'initial infectivity' was an excellent indicator of overall production of protein complexes. Further, we show that this initial infectivity can be mathematically described as a function of multiplicity of infection (MOI), correlating recombinant protein yield and virus titer. All these findings led us to develop the Titer Estimation for Quality Control (TEQC) method, which enables researchers to estimate initial infectivity, titer/MOI values in a simple and affordable way, and to use these values to quantitatively optimize protein expressions utilizing BEVS in a highly reproducible fashion.