• A polyphosphate kinase 1 (ppk1) mutant of Pseudomonas aeruginosa exhibits multiple ultrastructural and functional defects.

      Fraley, Cresson D.; Rashid, M. Harunur; Lee, Sam S. K.; Gottschalk, Rebecca; Harrison, Janine; Wood, Pauline J.; Brown, Michael R. W.; Kornberg, Arthur (National Academy of Sciences, 2007)
      Pseudomonas aeruginosa, of medical, environmental, and industrial importance, depends on inorganic polyphosphate (poly P) for a wide range of functions, especially survival. Mutants of PAO1 lacking poly P kinase 1, PPK1, the enzyme responsible for most poly P synthesis in Escherichia coli and other bacteria, are defective in motility, quorum sensing, biofilm formation, and virulence. We describe here multiple defects in the ppk1 mutant PAOM5, including a striking compaction of the nucleoid, distortion of the cell envelope, lack of planktonic motility and exopolymer production, and susceptibility to the beta-lactam antibiotic carbenicillin as well as desiccation. We propose that P. aeruginosa with reduced poly P levels undergoes ultrastructural changes that contribute to profound deficiencies in cellular functions.
    • Adenovirus vector-mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane.

      Cowen, Rachel L.; Williams, Judith C.; Emery, Steve; Blakey, David; Darling, John L.; Lowenstein, Pedro R.; Castro, Maria G. (nature.com, 2002)
      Carboxypeptidase G2 (CPG2) is a powerful prodrug-converting enzyme. Without a requirement for endogenous enzymes or cofactors, it can directly activate mustard alkylating prodrugs to cytotoxic species, killing both quiescent and dividing cells. This paper provides the first report of its use in the context of a clinically relevant delivery vehicle using adenovirus vectors. To strengthen the efficacy of the prodrug-activating system, the enzyme has been engineered to be secreted or glycosylphosphatidylinositol (GPI) anchored to the extracellular membrane of tumor cells, resulting in an enhanced bystander effect by facilitating diffusion of the active drug through extracellular, rather than intracellular, activation. Using the vectors, we have achieved expression of functional secreted or GPI-anchored CPG2 in a panel of tumor cell lines demonstrating no loss in efficacy as a result of GPI anchor retention. Despite variable transduction efficiencies inherent to these vectors, greater than 50% cell kill was achievable in all of the cell lines tested following only a single exposure to the prodrug ZD2767P. Even in cell lines refractive to infection with the vectors, substantial cell death was recorded, indicative of the enhanced bystander effect generated following extracellular prodrug activation. A direct evaluation of the efficacy of our system has been made against adenoviral delivery of herpes simples virus thymidine kinase plus ganciclovir (GCV), a suicide gene therapy approach already in the clinic. In a short-term human glioma culture (IN1760) resistant to the clinical chemotherapeutic drug CCNU (1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea), thymidine kinase/GCV effected no cell killing compared to 70% cell killing with our system.
    • Plasma membrane abundance of human aquaporin 5 is dynamically regulated by multiple pathways

      Kitchen, P; Öberg, F; Sjöhamn, J; Hedfalk, K; Bill, RM; Törnroth-Horsefield, S; Conner, Matthew T.; Conner, Alex C.; Molecular Organization and Assembly in Cells Doctoral Training Centre, University of Warwick, Coventry, United Kingdom. (Public Library of Science (PLoS), 2015-11-16)
      © 2015 Kitchen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Aquaporin membrane protein channels mediate cellular water flow. Human aquaporin 5 (AQP5) is highly expressed in the respiratory system and secretory glands where it facilitates the osmotically-driven generation of pulmonary secretions, saliva, sweat and tears. Dysfunctional trafficking of AQP5 has been implicated in several human disease states, including Sjögren's syndrome, bronchitis and cystic fibrosis. In order to investigate how the plasma membrane expression levels of AQP5 are regulated, we studied real-time translocation of GFP-tagged AQP5 in HEK293 cells. We show that AQP5 plasma membrane abundance in transfected HEK293 cells is rapidly and reversibly regulated by at least three independent mechanisms involving phosphorylation at Ser156, protein kinase A activity and extracellular tonicity. The crystal structure of a Ser156 phosphomimetic mutant indicates that its involvement in regulating AQP5 membrane abundance is not mediated by a conformational change of the carboxy-terminus. We suggest that together these pathways regulate cellular water flow.
    • The interaction between endogenous calcineurin and the plasma membrane calcium-dependent ATPase is isoform specific in breast cancer cells

      Holton, Marylouisa; Yang, Di; Wang, Weiguang; Mohamed, Tamer M. A.; Neyses, Ludwig; Armesilla, Angel (Elsevier, 2007)
      Plasma membrane calcium/calmodulin-dependent ATPases (PMCAs) are high affinity calcium pumps that extrude calcium from the cell. Emerging evidence suggests a novel role for PMCAs as regulators of calcium/calmodulin-dependent signal transduction pathways via interaction with specific partner proteins. In this work, we demonstrate that endogenous human PMCA2 and -4 both interact with the signal transduction phosphatase, calcineurin, whereas, no interaction was detected with PMCA1. The strongest interaction was observed between PMCA2 and calcineurin. The domain of PMCA2 involved in the interaction is equivalent to that reported for PMCA4b. PMCA2-calcineurin interaction results in inhibition of the calcineurin/nuclear factor of activated T-cells signalling pathway.