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Lexington, KY, United States

Ortega J.,University of Kentucky | Li J.Y.,Paul Laurence Dunbar High School | Li J.Y.,Yale University | Lee S.,University of Kentucky | And 5 more authors.
Proceedings of the National Academy of Sciences of the United States of America | Year: 2015

Proliferating or cell nuclear antigen (PCNA) plays essential roles in eukaryotic cells during DNA replication, DNA mismatch repair (MMR), and other events at the replication fork. Earlier studies show that PCNA is regulated by posttranslational modifications, including phosphorylation of tyrosine 211 (Y211) by the epidermal growth factor receptor (EGFR). However, the functional significance of Y211-phosphorylated PCNA remains unknown. Here, we show that PCNA phosphorylation by EGFR alters its interaction with mismatch-recognition proteins MutSα and MutSβ and interferes with PCNA-dependent activation of MutLα endonuclease, thereby inhibiting MMR at the initiation step. Evidence is also provided that Y211-phosphorylated PCNA induces nucleotide misincorporation during DNA synthesis. These findings reveal a novel mechanism by which Y211-phosphorylated PCNA promotes cancer development and progression via facilitating error-prone DNA replication and suppressing the MMR function. © 2015, National Academy of Sciences. All rights reserved. Source

Sun W.,University of Kentucky | Yang K.,Paul Laurence Dunbar High School | Yang F.,University of Kentucky
Journal of Polymer Research | Year: 2015

A simple template consisting of two contacting metallic wires was developed to construct surface structures on PMMA films. Using this template, surface structures including arcs and straight stripes were formed on the surface of PMMA films via the evaporation of toluene droplets. The effect of the angle between two wires and the film thickness on the geometric characteristics of the structures formed was studied. For the same film thickness, the amplitude is approximately a linear, increasing function of the wavelength with approximately the same slope, independent of the angle between the wires. The wavelength of the surface structures increases with the film thickness for the same distance to the wire and the same angle between the wires. This technique provides a viable technique to form surface structures over a large surface area of polymer films for various applications. © 2015, Springer Science+Business Media Dordrecht. Source

Li X.,University of Kentucky | Stevens P.D.,University of Kentucky | Liu J.,University of Kentucky | Yang H.,Taizhou Hospital | And 7 more authors.
Gastroenterology | Year: 2014

Background & Aims Hyperactivation of the RAS-RAF signaling pathway in colorectal tumors is associated with metastasis and poor outcomes of patients. Little is known about how RAS-RAF signaling is turned off once activated. We investigated how the pH domain and leucine-rich repeat protein phosphatases (PHLPPs) control RAS-RAF signaling and colorectal cancer (CRC) development. Methods We used co-immunoprecipitation assays to identify substrates of PHLPP1 and PHLPP2. We studied phosphorylation of RAF1 in CRC cells that express exogenous PHLPP1 or PHLPP2, or lentiviral-based small hairpin RNAs against their transcripts; we measured effects on cell motility, migration, and invasion in vitro. Tumor progression and survival were analyzed in Phlpp1-/- ApcMin and ApcMin/Phlpp1-/- mice. Microarray datasets of colorectal tumor and nontumor tissues were analyzed for PHLPP gene expression. Results PHLPP1 and 2 were found to dephosphorylate RAF1 at S338, inhibiting its kinase activity in vitro and in CRC cells. In cells, knockdown of PHLPP1 or PHLPP2 increased the amplitude and duration of RAF-MEK-ERK signaling downstream of epidermal growth factor receptor and KRAS, whereas overexpression had the opposite effect. In addition, knockdown of PHLPP1 or PHLPP2 caused CRC cells to express markers of the epithelial-mesenchymal transition, and increased cell migration and invasion. ApcMin/Phlpp1-/- mice had decreased survival and developed larger intestinal and colon tumors compared to ApcMin mice. Whereas ApcMin mice developed mostly low-grade adenomas, 20% of the tumors that developed in ApcMin/ Phlpp1-/- mice were invasive adenocarcinomas. Normal villi and adenomas of ApcMin/Phlpp1-/- mice had significantly fewer apoptotic cells than ApcMin mice. Human CRC patient microarray data revealed that the expression of PHLPP1 or PHLPP2 is positively correlated with CDH1. Conclusions PHLPP1 and PHLPP2 dephosphorylate RAF1 to reduce its signaling, increase the invasive and migratory activities of CRC cells, and activate the epithelial-mesenchymal transition. In ApcMin mice, loss of PHLPP1 promotes tumor progression. Source

Fang J.,Zhejiang University | Yu L.,University of Kentucky | Wu M.,Paul Laurence Dunbar High School | Wei Y.,University of Kentucky
Journal of Biomolecular Structure and Dynamics | Year: 2013

The resistance-nodulation-cell division family multidrug transporter Acriflavine resistance protein B (AcrB) from Eschericha coli is an obligate homotrimer. Approximately, 45% of the inter-subunit interface is contributed by a protruding loop (also referred to as the thumb) and its corresponding binding tunnel in the neighboring subunit. In an earlier study, we have demonstrated that a single Pro to Gly mutation in the loop drastically destabilized AcrB trimer and reduced its substrate efflux activity. To further dissect the role of the loop during AcrB trimerization, we performed Ala scanning of the loop and examined the effect of each mutation on protein activity. We found that not all conserved residues are important for AcrB function and likewise not all critical residues are conserved. In addition, we replaced the loop of AcrB with the loop of MexB, which is a highly conserved homolog of AcrB. The resultant chimeric protein remained partly active. Structural characterization of the chimeric protein indicated that it was well folded and existed as a mixture of monomer and trimer. Our results indicate that the loop to tunnel interaction, while critical to trimerization and efflux function, is in general rather flexible and tolerant to mutations. In addition, all mutations in the loop that resulted in reduced function clustered closely, suggesting that this may be the site of inter-subunit recognition during trimerization and/or a locking zone to stabilize the inter-subunit interaction during trimerization. Copyright © 2012 Taylor & Francis. Source

Knecht L.D.,University of Miami | Ali N.,Paul Laurence Dunbar High School | Wei Y.,University of Kentucky | Hilt J.Z.,University of Kentucky | Daunert S.,University of Miami
ACS Nano | Year: 2012

Nanomaterials have found numerous applications as tunable, remotely controlled platforms for drug delivery, hyperthermia cancer treatment, and various other biomedical applications. The basis for the interest lies in their unique properties achieved at the nanoscale that can be accessed via remote stimuli. These properties could then be exploited to simultaneously activate secondary systems that are not remotely actuatable. In this work, iron oxide nanoparticles are encapsulated in a bisacrylamide cross-linked polyacrylamide hydrogel network along with a model dehalogenase enzyme, L-2-HAD ST. This thermophilic enzyme is activated at elevated temperatures and has been shown to have optimal activity at 70 °C. By exposing the Fe 3O 4 nanoparticles to a remote stimulus, an alternating magnetic field (AMF), enhanced system heating can be achieved, thus remotely activating the enzyme. The internal heating of the nanocomposite hydrogel network in the AMF results in a 2-fold increase in enzymatic activity as compared to the same hydrogel heated externally in a water bath, suggesting that the internal heating of the nanoparticles is more efficient than the diffusion-limited heating of the water bath. This system may prove useful for remote actuation of biomedical and environmentally relevant enzymes and find applications in a variety of fields. © 2012 American Chemical Society. Source

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