Browsing by Author "Winton, Carly E."

Now showing 1 - 4 of 4
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    A microchip platform for structural oncology applications
    Winton, Carly E.; Gilmore, Brian L.; Demmert, Andrew C.; Karageorge, Vasilea; Sheng, Zhi; Kelly, Deborah F. (2016)
    Recent advances in the development of functional materials offer new tools to dissect human health and disease mechanisms. The use of tunable surfaces is especially appealing as substrates can be tailored to fit applications involving specific cell types or tissues. Here we use tunable materials to facilitate the three-dimensional (3D) analysis of BRCA1 gene regulatory complexes derived from human cancer cells. We employed a recently developed microchip platform to isolate BRCA1 protein assemblies natively formed in breast cancer cells with and without BRCA1 mutations. The captured assemblies proved amenable to cryo-electron microscopy (EM) imaging and downstream computational analysis. Resulting 3D structures reveal the manner in which wild-type BRCA1 engages the RNA polymerase II (RNAP II) core complex that contained K63-linked ubiquitin moieties-a putative signal for DNA repair. Importantly, we also determined that molecular assemblies harboring the BRCA1(5382insC) mutation exhibited altered protein interactions and ubiquitination patterns compared to wild-type complexes. Overall, our analyses proved optimal for developing new structural oncology applications involving patient-derived cancer cells, while expanding our knowledge of BRCA1's role in gene regulatory events.
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    Molecular Analysis of BRCA1 in Human Breast Cancer Cells Under Oxidative Stress
    Gilmore, Brian L.; Liang, Yanping; Winton, Carly E.; Patel, Kaya; Karageorge, Vasilea; Varano, A. Cameron; Dearnaley, William J.; Sheng, Zhi; Kelly, Deborah F. (Nature Publishing Group, 2017-03-06)
    The precise manner in which physical changes to the breast cancer susceptibility protein (BRCA1) affect its role in DNA repair events remain unclear. Indeed, cancer cells harboring mutations in BRCA1 suffer from genomic instability and increased DNA lesions. Here, we used a combination of molecular imaging and biochemical tools to study the properties of the BRCA1 in human cancer cells. Our results reveal new information for the manner in which full-length BRCA1 engages its binding partner, the BRCA1-associated Ring Domain protein (BARD1) under oxidative stress conditions. We also show how physical differences between wild type and mutated BRCA15382insC impact the cell’s response to oxidative damage. Overall, we demonstrate how clinically relevant changes to BRCA1 affect its structure-function relationship in hereditary breast cancer.
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    A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of Human Disease
    Gilmore, Brian L.; Winton, Carly E.; Demmert, Andrew C.; Tanner, Justin R.; Bowman, Sam; Karageorge, Vasilea; Patel, Kaya; Sheng, Zhi; Kelly, Deborah F. (Springer Nature, 2015-09-23)
    We present a new molecular toolkit to investigate protein assemblies natively formed in the context of human disease. The system employs tunable microchips that can be decorated with switchable adaptor molecules to select for target proteins of interest and analyze them using molecular microscopy. Implementing our new streamlined microchip approach, we could directly visualize BRCA1 gene regulatory complexes from patient-derived cancer cells for the first time.
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    Structural analysis of BRCA1 reveals modification hotspot
    Liang, Yanping; Dearnaley, William J.; Varano, A. Cameron; Winton, Carly E.; Gilmore, Brian L.; Alden, Nick A.; Sheng, Zhi; Kelly, Deborah F. (American Association for the Advancement of Science, 2017-09-01)