Browsing by Author "Kelly, Deborah F."
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- 3-D Bio-inspired Microenvironments for In Vitro Cell MigrationHosseini, Seyed Yahya (Virginia Tech, 2015-10-21)Cancer metastasis is the leading cause of death related to cancer diseases. Once the cancer cells depart the primary tumor site and enter the blood circulation, they spread through the body and will likely initiate a new tumor site. Therefore, understanding the cell migration and stopping the spread in the initial stage is the utmost of importance. In this dissertation, we have proposed a 3-D microenvironment that (partially) mimics the structures, complexity and circulation of human organs for cell migration studies. We have developed the tools to fabricate 3-D complex geometries in PDMS from our previously developed single-mask, single-etch technology in silicon. In this work, 3-D patterns are transferred from silicon structures to glass following anodic bonding and high temperature glass re-flow processes. Silicon is etched back thoroughly via wet etching and the glass is used as master device to create 3-D PDMS structures for use in dielectrophoresis cell sorting applications. Furthermore, this work has been modified to fabricate 3-D master devices in PDMS to create 3-D structures in collagen hydrogels to mimic native tissue structures. We have studied the interaction of endothelial cells with model geometries of blood vessels in collagen hydrogel at different concentrations to mimic the biomechanical properties of tissues varying from normal to tumor under the growth factor stimulation. Finally, we have designed and fabricated a silicon-based transmigration well with a 30um-thick membrane and 8um pores. This platform includes a deep microfluidic channel on the back-side sealed with a glass wafer. The migratory behavior of highly metastatic breast cancer cells, MDA-MB-231, is tested under different drug treatment conditions. This versatile platform will enable the application of more complex fluidic circulation profile, enhanced integration with other technologies, and running multiple assays simultaneously.
- Binding properties of adaptor proteins Tollip and Tom1Brannon, Mary Katherine (Virginia Tech, 2015-07-02)Adaptor proteins, like Tollip and Tom1, facilitate cellular cargo sorting through their ubiquitin-binding domains. Tollip and Tom1 bind to each other through their TBD and GAT domains, respectively, whereas Tollip interacts with phosphatidylinositol-3-phosphate (PtdIns(3)P)-containing endosomal membranes. Tom1 and Tollip interaction and association with endosomes is proposed to be involved in the lysosomal degradation of polyubiquitinated cargo. Through cellular, biochemical, and biophysical techniques, we have further characterized the association of Tom1 with Tollip. Mutations in the binding interface of the Tom1 GAT and Tollip TBD complex leads to a subcellular mis-localization of both proteins, indicating that Tom1 may serve to direct Tollip to specific cellular pathways. It was determined that Tom1 inhibits the binding of Tollip to PtdIns(3)P and inhibition was reversed when mutations in the binding interface of the Tom1 GAT and Tollip TBD were present. Furthermore, it was established that, upon the binding of Tollip TBD to Tom1 GAT, ubiquitin is inhibited from binding to Tom1 GAT. It was also demonstrated that Tom1 GAT, but not Tollip TBD, can weakly bind to PtdIns(3)P. Consequently, we propose that association of Tom1 may serve to direct Tollip for involvement in specific cell signaling pathways. Gaining insight into the function of Tom1 and Tollip may lead to their use as therapeutic targets for increasing the efficiency of cargo trafficking and also for patients recovering from various cardiac injuries.
- Bioimpedance spectroscopy of breast cancer cells: A microsystems approachSrinivasaraghavan, Vaishnavi (Virginia Tech, 2015-11-04)Bioimpedance presents a versatile, label-free means of monitoring biological cells and their responses to physical, chemical and biological stimuli. Breast cancer is the second most common type of cancer among women in the United States. Although significant progress has been made in diagnosis and treatment of this disease, there is a need for robust, easy-to-use technologies that can be used for the identification and discrimination of critical subtypes of breast cancer in biopsies obtained from patients. This dissertation makes contributions in three major areas towards addressing the goal. First, we developed miniaturized bioimpedance sensors using MEMS and microfluidics technology that have the requisite traits for clinical use including reliability, ease-of-use, low-cost and disposability. Here, we designed and fabricated two types of bioimpedance sensors. One was based on electric cell-substrate impedance sensing (ECIS) to monitor cell adhesion based events and the other was a microfluidic device with integrated microelectrodes to examine the biophysical properties of single cells. Second, we examined a panel of triple negative breast cancer (TNBC) cell lines and a hormone therapy resistant model of breast cancer in order to improve our understanding of the bioimpedance spectra of breast cancer subtypes. Third, we explored strategies to improve the sensitivity of the microelectrodes to bioimpedance measurements from breast cancer cells. We investigated nano-scale coatings on the surface of the electrode and geometrical variations in a branched electrode design to accomplish this. This work demonstrates the promise of bioimpedance technologies in monitoring diseased cells and their responses to pharmaceutical agents, and motivates further research in customization of this technique for use in personalized medicine.
- Capturing Enveloped Viruses on Affinity Grids for Downstream Cryo- Electron Microscopy ApplicationsKiss, G.; Chen, X. M.; Brindley, M. A.; Campbell, P.; Afonso, C. L.; Ke, Z. L.; Holl, J. M.; Guerrero-Ferreira, R. C.; Byrd-Leotis, L. A.; Steel, J.; Steinhauer, D. A.; Plemper, R. K.; Kelly, Deborah F.; Spearman, P. W.; Wright, E. R. (Cambridge University Press, 2014-02)Electron microscopy (EM), cryo-electron microscopy (cryo-EM), and cryo-electron tomography (cryo-ET) are essential techniques used for characterizing basic virus morphology and determining the three-dimensional structure of viruses. Enveloped viruses, which contain an outer lipoprotein coat, constitute the largest group of pathogenic viruses to humans. The purification of enveloped viruses from cell culture presents certain challenges. Specifically, the inclusion of host-membrane-derived vesicles, the complete destruction of the viruses, and the disruption of the internal architecture of individual virus particles. Here, we present a strategy for capturing enveloped viruses on affinity grids (AG) for use in both conventional EM and cryo-EM/ET applications. We examined the utility of AG for the selective capture of human immunodeficiency virus virus-like particles, influenza A, and measles virus. We applied nickel-nitrilotriacetic acid lipid layers in combination with molecular adaptors to selectively adhere the viruses to the AG surface. This further development of the AG method may prove essential for the gentle and selective purification of enveloped viruses directly onto EM grids for ultrastructural analyses.
- Casein Kinase 1 Epsilon Regulates Glioblastoma Cell SurvivalVarghese, Robin T.; Young, Sarah; Pham, Lily; Liang, Yanping; Pridham, Kevin J.; Guo, Sujuan; Murphy, Susan F.; Kelly, Deborah F.; Sheng, Zhi (Nature, 2018-09-11)Glioblastoma is the most common malignant brain cancer with a dismal prognosis. The difficulty in treating glioblastoma is largely attributed to the lack of effective therapeutic targets. In our previous work, we identified casein kinase 1 ε (CK1ε, also known as CSNK1E) as a potential survival factor in glioblastoma. However, how CK1ε controls cell survival remains elusive and whether targeting CK1ε is a possible treatment for glioblastoma requires further investigation. Here we report that CK1ε was expressed at the highest level among six CK1 isoforms in glioblastoma and enriched in high-grade glioma, but not glia cells. Depletion of CK1ε remarkably inhibited the growth of glioblastoma cells and suppressed self-renewal of glioblastoma stem cells, while having limited effect on astrocytes. CK1ε deprivation activated β-catenin and induced apoptosis, which was further counteracted by knockdown of β-catenin. The CK1ε inhibitor IC261, but not PF-4800567, activated β-catenin and blocked the growth of glioblastoma cells and glioblastoma stem cells. Congruently, IC261 elicited a robust growth inhibition of human glioblastoma xenografts in mice. Together, our results demonstrate that CK1ε regulates the survival of glioblastoma cells and glioblastoma stem cells through β-catenin signaling, underscoring the importance of targeting CK1ε as an effective treatment for glioblastoma.
- Connexin 43 confers chemoresistance through activating PI3KPridham, Kevin J.; Shah, Farah; Hutchings, Kasen R.; Sheng, Kevin L.; Guo, Sujuan; Liu, Min; Kanabur, Pratik; Lamouille, Samy Y.; Lewis, Gabrielle; Morales, Marc; Jourdan, L. Jane; Grek, Christina L.; Ghatnekar, Gautam S.; Varghese, Robin T.; Kelly, Deborah F.; Gourdie, Robert G.; Sheng, Zhi (Springer Nature, 2022-01-12)Circumventing chemoresistance is crucial for effectively treating cancer including glioblastoma, a lethal brain cancer. The gap junction protein connexin 43 (Cx43) renders glioblastoma resistant to chemotherapy; however, targeting Cx43 is difficult because mechanisms underlying Cx43-mediated chemoresistance remain elusive. Here we report that Cx43, but not other connexins, is highly expressed in a subpopulation of glioblastoma and Cx43 mRNA levels strongly correlate with poor prognosis and chemoresistance in this population, making Cx43 the prime therapeutic target among all connexins. Depleting Cx43 or treating cells with αCT1–a Cx43 peptide inhibitor that sensitizes glioblastoma to the chemotherapy temozolomide–inactivates phosphatidylinositol-3 kinase (PI3K), whereas overexpression of Cx43 activates this signaling. Moreover, αCT1-induced chemo-sensitization is counteracted by a PI3K active mutant. Further research reveals that αCT1 inactivates PI3K without blocking the release of PI3K-activating molecules from membrane channels and that Cx43 selectively binds to the PI3K catalytic subunit β (PIK3CB, also called PI3Kβ or p110β), suggesting that Cx43 activates PIK3CB/p110β independent of its channel functions. To explore the therapeutic potential of simultaneously targeting Cx43 and PIK3CB/p110β, αCT1 is combined with TGX-221 or GSK2636771, two PIK3CB/p110β-selective inhibitors. These two different treatments synergistically inactivate PI3K and sensitize glioblastoma cells to temozolomide in vitro and in vivo. Our study has revealed novel mechanistic insights into Cx43/PI3K-mediated temozolomide resistance in glioblastoma and demonstrated that targeting Cx43 and PIK3CB/p110β together is an effective therapeutic approach for overcoming chemoresistance.
- High-Resolution Imaging of Human Cancer Proteins Using Microprocessor MaterialsSolares, Maria J.; Jonaid, G. M.; Luqiu, William Y.; Berry, Samantha; Khadela, Janki; Liang, Yanping; Evans, Madison C.; Pridham, Kevin J.; Dearnaley, William J.; Sheng, Zhi; Kelly, Deborah F. (Wiley-V C H , 2022-07-14)Mutations in tumor suppressor genes, such as Tumor Protein 53 (TP53), are heavily implicated in aggressive cancers giving rise to gain- and loss-of-function phenotypes. While individual domains of the p53 protein have been studied extensively, structural information for full-length p53 remains incomplete. Functionalized microprocessor chips (microchips) with properties amenable to electron microscopy permitted us to visualize complete p53 assemblies for the first time. The new structures revealed p53 in an inactive dimeric state independent of DNA binding. Residues located at the protein-protein interface corresponded with modification sites in cancer-related hot spots. Changes in these regions may amplify the toxic effects of clinical mutations. Taken together, these results contribute advances in technology and imaging approaches to decode native protein models in different states of activation.
- A microchip platform for structural oncology applicationsWinton, 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.
- Microfluidic Approaches for Probing Protein Phosphorylation in CellsDeng, Jingren (Virginia Tech, 2018-07-31)Protein phosphorylation plays critical roles in diverse cellular functions, including cell cycle, growth, differentiation, and apoptosis. Deregulated phospho-signaling is often associated with many human diseases and cancers. Despite tremendous efforts to investigate the molecular mechanisms that control the functionality of phospho-signaling pathways, only limited progress has been made on describing the temporal and spatial profiles of cellular protein phosphorylation. The main challenges associated with the study of phospho-signaling processes in cells are related to the short time-scale of certain phosphorylation and dephosphorylation events, the low abundance of the phosphorylated protein forms as compared to their non-phosphorylated counterparts, the complicated and time-consuming sample preparation methods that are accompanying such type of work, and, last, the performance of the detection methods that are suitable for assessing protein phosphorylation. To tackle the challenges associated with the investigation of protein phosphorylation in cells, our objective was to develop a combined mass spectrometry (MS) and microfluidics strategy that enables fast sampling and sensitive detection of key signaling phosphoproteins in complex biological samples. MS is the most widely used analytical tool in the field of proteomics due to its high sensitivity, specificity, and throughput. Microfluidics has been proven as a suitable platform for handling small volumes of scarce samples, being also amenable to automation, integration, and multiplexing. To achieve our objective, this study was conducted in multiple steps: (1) We performed a comprehensive analysis of the factors that affect the performance of mass spectrometry detection (i.e., sensitivity, reproducibility, ability to accurately identify a large number of proteins from complex samples), when used in conjunction with technologies that are conducted in a non-standard fashion, on short time-scales; (2) We developed and evaluated a miniaturized strategy for rapid proteolytic digestion and phosphopeptide enrichment; (3) We demonstrated sensitive detection and quantification of phosphopeptides from complex biological samples using multiple reaction monitoring mass spectrometry (MRM-MS) and microfluidic sample processing; and (4) We developed a microfluidic platform for handling and processing cells that enables the investigation of biological processes that occur on short time-scales, and that can be integrated with the devices developed for the analysis of phospho-proteins. SKBR3 cells were used as a model system for developing and demonstrating the microfluidic chips. The detection and quantification of phospho-proteins involved in MAPK (mitogen activated protein kinase) signaling pathways was achieved at the low nM level. Overall, this study demonstrates proof-of-concept applicability of a microfluidics-MS strategy for monitoring phosphorylation processes in signaling networks.
- Molecular Analysis of BRCA1 in Human Breast Cancer Cells Under Oxidative StressGilmore, 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.
- Molecular structure and dimeric organization of the Notch extracellular domain as revealed by electron microscopyKelly, Deborah F.; Lake, Robert J.; Middelkoop, Teije C.; Fan, Hua-Ying; Artavanis-Tsakonas, Spyros; Walz, Thomas (2010-05-07)BACKGROUND: The Notch receptor links cell fate decisions of one cell to that of the immediate cellular neighbor. In humans, malfunction of Notch signaling results in diseases and congenital disorders. Structural information is essential for gaining insight into the mechanism of the receptor as well as for potentially interfering with its function for therapeutic purposes. METHODOLOGY/PRINCIPAL FINDINGS: We used the Affinity Grid approach to prepare specimens of the Notch extracellular domain (NECD) of the Drosophila Notch and human Notch1 receptors suitable for analysis by electron microscopy and three-dimensional (3D) image reconstruction. The resulting 3D density maps reveal that the NECD structure is conserved across species. We show that the NECD forms a dimer and adopts different yet defined conformations, and we identify the membrane-proximal region of the receptor and its ligand-binding site. CONCLUSIONS/SIGNIFICANCE: Our results provide direct and unambiguous evidence that the NECD forms a dimer. Our studies further show that the NECD adopts at least three distinct conformations that are likely related to different functional states of the receptor. These findings open the way to now correlate mutations in the NECD with its oligomeric state and conformation.
- Molecular Surveillance of Viral Processes Using Silicon Nitride MembranesGilmore, Brian L.; Tanner, Justin R.; McKell, Allison O.; Boudreaux, Crystal E.; Dukes, Madeline J.; McDonald, Sarah M.; Kelly, Deborah F. (MDPI, 2013-03)Here we present new applications for silicon nitride (SiN) membranes to evaluate biological processes. We determined that 50-nanometer thin films of SiN produced from silicon wafers were sufficiently durable to bind active rotavirus assemblies. A direct comparison of SiN microchips with conventional carbon support films indicated that SiN performs equivalent to the traditional substrate to prepare samples for Electron Microscopy (EM) imaging. Likewise, SiN films coated with Ni-NTA affinity layers concentrated rotavirus particles similarly to affinity-coated carbon films. However, affinity-coated SiN membranes outperformed glow-discharged conventional carbon films 5-fold as indicated by the number of viral particles quantified in EM images. In addition, we were able to recapitulate viral uncoating and transcription mechanisms directed onto the microchip surfaces. EM images of these processes revealed the production of RNA transcripts emerging from active rotavirus complexes. These results were confirmed by the functional incorporation of radiolabeled nucleotides into the nascent RNA transcripts. Collectively, we demonstrate new uses for SiN membranes to perform molecular surveillance on life processes in real-time.
- A Molecular Toolkit to Visualize Native Protein Assemblies in the Context of Human DiseaseGilmore, 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.
- Patient-derived glioblastoma stem cells respond differentially to targeted therapiesKanabur, Pratik; Guo, Sujuan; Simonds, Gary S.; Kelly, Deborah F.; Gourdie, Robert G.; Verbridge, Scott S.; Sheng, Zhi (Impact Journals, 2016-12-27)The dismal prognosis of glioblastoma is, at least in part, attributable to the difficulty in eradicating glioblastoma stem cells (GSCs). However, whether this difficulty is caused by the differential responses of GSCs to drugs remains to be determined. To address this, we isolated and characterized ten GSC lines from established cell lines, xenografts, or patient specimens. Six lines formed spheres in a regular culture condition, whereas the remaining four lines grew as monolayer. These adherent lines formed spheres only in plates coated with poly-2-hydroxyethyl methacrylate. The self-renewal capabilities of GSCs varied, with the cell density needed for sphere formation ranging from 4 to 23.8 cells/well. Moreover, a single non-adherent GSC either remained quiescent or divided into two cells in four-seven days. The stem cell identity of GSCs was further verified by the expression of nestin or glial fibrillary acidic protein. Of the two GSC lines that were injected in immunodeficient mice, only one line formed a tumor in two months. The protein levels of NOTCH1 and platelet derived growth factor receptor alpha positively correlated with the responsiveness of GSCs to γ-secretase inhibitor IX or imatinib, two compounds that inhibit these two proteins, respectively. Furthermore, a combination of temozolomide and a connexin 43 inhibitor robustly inhibited the growth of GSCs. Collectively, our results demonstrate that patient-derived GSCs exhibit different growth rates in culture, possess differential capabilities to form a tumor, and have varied responses to targeted therapies. Our findings underscore the importance of patient-derived GSCs in glioblastoma research and therapeutic development.
- A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applicationsGuo, Sujuan; Liang, Yanping; Murphy, Susan F.; Huang, Angela; Shen, Haihong; Kelly, Deborah F.; Sobrado, Pablo; Sheng, Zhi (Taylor & Francis, 2015-03-01)The lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagy-modulating drugs.
- Revealing Molecular Adversaries of Human Health Using Advanced Imaging TechnologyVarano, Ann Cameron (Virginia Tech, 2018-12-07)Single particle electron microscopy (EM) allows us to examine the molecular world and gain insights into protein structures implicated in human disease. Visualizing the 3D architecture of the macromolecules can inform drug design and preventative care. While X-ray crystallography and NMR are able to resolve atomic structures, the methodology is better suited for smaller structures with limited flexibility. Single particle EM allows us analyze larger structures that have inherent flexibility. Protein structures can broadly be categorized as symmetry or asymmetric. There are computational advantages when analyzing symmetrical structures. Specifically, structural information can be extrapolated from fewer vantage points. Thus, symmetrical macromolecules are an advantageous for pioneering new methodologies in single particle EM. Rotavirus double layered particles (DLPs) are large macromolecular complexes that display icosahedral symmetry. Previous studies have led to a high resolution structure of transcriptionally inactive rotavirus frozen in time. However, to more fully understand rotavirus we need to examine the structure under transcriptionally active conditions. To expand our understanding, we first evaluated these viral assemblies using cryo-EM under active and inactive conditions. We found both internal and external structural differences. Based on these findings we sought to further our understanding of these nano-machines by developing a liquid cell environment to evaluate their dynamics over time. Our research not only developed a new methodology to evaluate active particles over time, we also found that the mobility of the DLPs were directly correlated to the level of transcriptional activity. When analyzing asymmetrical and flexible protein complexes previous studies have utilized methodologies to limit the proteins' conformational variability. While this does allow for a higher resolution structure, it limits our understanding to a specific orientation and compromises the biological insights. BRCA1 is an asymmetric protein containing a large flexible region and is important in the prevention of breast cancer. We utilize silicon nitride microchips with integrated wells and decorated with a lipid monolayer to capture and image BRCA1 complexes. This imaging platform minimizes heterogeneity and ensures the sample quality while not biasing confirmation. Thus, allowing for high resolution cryo-EM imaging of flexible native proteins. We were able to examine BRCA1 complexes from cells at both the primary and metastatic sites. Our ability to visualize these proteins in their native form provide insights into the variability of BRCA1 in disease progression. We found that BRCA1 complexes isolated from metastatic cells have additional density in the C-terminal domain. Our data suggests this density it due an interaction with p53. Overall, our methodologies highlight the power of single particle EM for studying protein complexes. Furthermore, our findings emphasize the importance of examining protein complexes in their native state.
- Selective regulation of chemosensitivity in glioblastoma by phosphatidylinositol 3-kinase betaPridham, Kevin J.; Hutchings, Kasen R.; Beck, Patrick; Liu, Min; Xu, Eileen; Saechin, Erin; Bui, Vincent; Patel, Chinkal; Solis, Jamie; Huang, Leah; Tegge, Allison; Kelly, Deborah F.; Sheng, Zhi (Elsevier, 2024-06-21)Resistance to chemotherapies such as temozolomide is a major hurdle to effectively treat therapy-resistant glioblastoma. This challenge arises from the activation of phosphatidylinositol 3-kinase (PI3K), which makes it an appealing therapeutic target. However, non-selectively blocking PI3K kinases PI3K⍺/β/𝛿/𝛾 has yielded undesired clinical outcomes. It is, therefore, imperative to investigate individual kinases in glioblastoma’s chemosensitivity. Here,wereport that PI3K kinases were unequally expressed in glioblastoma, with levels of PI3Kβ being the highest. Patients deficient of O6-methylguanine-DNA-methyltransferase(MGMT) and expressing elevated levels of PI3Kβ, defined as MGMT-deficient/PI3Kβ-high, were less responsive to temozolomide and experienced poor prognosis. Consistently, MGMT-deficient/PI3Kβ-high glioblastoma cells were resistant to temozolomide. Perturbation of PI3Kβ, but not other kinases, sensitized MGMTdeficient/ PI3Kβ-high glioblastoma cells or tumors to temozolomide. Moreover, PI3Kβ-selective inhibitors and temozolomide synergistically mitigated the growth of glioblastoma stem cells. Our results have demonstrated an essential role of PI3Kβ in chemoresistance, making PI3Kβ-selective blockade an effective chemosensitizer for glioblastoma.
- Structural analysis of BRCA1 reveals modification hotspotLiang, 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)
- A Structural Approach to Unveil the Role of BRCA1 in the Context of TranscriptionWinton, Carly Elizabeth (Virginia Tech, 2016-01-19)The research presented in this thesis aims to uncover the intricate manner in which BRCA1 interacts with RNAP II during mRNA production utilizing a unique microchip system developed in our lab. We were first able to prove the effectiveness of our tunable system using a breast cancer model of patient derived triple negative breast cancer (TNBC) cells. Here we switched out different mammalian antibodies and collected images of the same structure from different angles. This served many purposes: (1) it proved the system could be tuned for specific uses; (2) it demonstrated that all subunits were present in the complex; (3) it eliminated the need for the tilt function allowing for a less intensive computational procedure. In the BRCA1 wild type cell line we were able to incorporate into our 3D reconstruction, atomic models of the BRCA1-BARD1 heterodimer and the RNAP II core in regions of major unoccupied density. Other areas of minor missing density were overlaid with a short strand of DNA and ubiquitin moieties, which proved agreement with Co-IPs. Next we sought to compare the wild type structure with a BRCA1 mutant variety. Using these techniques, we determined the 3D structure of the mutated complex. After further analysis slight differences were detected between the two complexes, especially in the placement of the atomic models. Overall we were able to determine structural abnormalities that occur when a mutation is present in BRCA1 that may have future applications for targetable therapy in TNBC patients. Moreover, by using TNBC as the disease model, we have created a platform that can be used to evaluate other human diseases due to the tunable nature of our microchip system.
- Survival kinase genes present prognostic significance in glioblastomaVarghese, Robin T.; Liang, Yanping; Guan, Ting; Franck, Christopher T.; Kelly, Deborah F.; Sheng, Zhi (Impact Journals, 2016-04-12)Cancer biomarkers with a strong predictive power for diagnosis/prognosis and a potential to be therapeutic targets have not yet been fully established. Here we employed a loss-of-function screen in glioblastoma (GBM), an infiltrative brain tumor with a dismal prognosis, and identified 20 survival kinase genes (SKGs). Survival analyses using The Cancer Genome Atlas (TCGA) datasets revealed that the expression of CDCP1, CDKL5, CSNK1E, IRAK3, LATS2, PRKAA1, STK3, TBRG4, and ULK4 stratified GBM prognosis with or without temozolomide (TMZ) treatment as a covariate. For the first time, we found that GBM patients with a high level of NEK9 and PIK3CB had a greater chance of having recurrent tumors. The expression of CDCP1, IGF2R, IRAK3, LATS2, PIK3CB, ULK4, or VRK1 in primary GBM tumors was associated with recurrence-related prognosis. Notably, the level of PIK3CB in recurrent tumors was much higher than that in newly diagnosed ones. Congruent with these results, genes in the PI3K/AKT pathway showed a significantly strong correlation with recurrence rate, further highlighting the pivotal role of PIK3CB in the disease progression. Importantly, 17 SKGs together presented a novel GBM prognostic signature. SKGs identified herein are associated with recurrence rate and present prognostic significance in GBM, thereby becoming attractive therapeutic targets.