A fission yeast platform for heterologous expression of mammalian adenylyl cyclases and high throughput screening.

Autor: Getz RA; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA., Kwak G; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA., Cornell S; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA., Mbugua S; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA. Electronic address: mbugua@bc.edu., Eberhard J; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA. Electronic address: eberharj@bc.edu., Huang SX; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA. Electronic address: huangsi@bc.edu., Abbasi Z; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA. Electronic address: abbasiz@bc.edu., de Medeiros AS; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA., Thomas R; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA. Electronic address: thomasry@bc.edu., Bukowski B; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA. Electronic address: bukowskb@bc.edu., Dranchak PK; National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA. Electronic address: patricia.dranchak@nih.gov., Inglese J; National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, MD 20850, USA. Electronic address: jinglese@mail.nih.gov., Hoffman CS; Biology Department, Boston College, 140 Commonwealth Ave., Chestnut Hill, MA 02467, USA. Electronic address: hoffmacs@bc.edu.
Jazyk: angličtina
Zdroj: Cellular signalling [Cell Signal] 2019 Aug; Vol. 60, pp. 114-121. Date of Electronic Publication: 2019 Apr 24.
DOI: 10.1016/j.cellsig.2019.04.010
Abstrakt: The fission yeast Schizosaccharomyces pombe uses a cAMP signaling pathway to link glucose-sensing to Protein Kinase A activity in order to regulate cell growth, sexual development, gluconeogenesis, and exit from stationary phase. We previously used a PKA-repressed fbp1-ura4 reporter to conduct high throughput screens (HTSs) for inhibitors of heterologously-expressed mammalian cyclic nucleotide phosphodiesterases (PDEs). Here, we describe the successful expression of all ten mammalian adenylyl cyclase (AC) genes, along with the human GNAS Gα s gene. By measuring expression of an fbp1-GFP reporter together with direct measurements of intracellular cAMP levels, we can detect both basal AC activity from all ten AC genes as well as GNAS-stimulated activity from eight of the nine transmembrane ACs (tmACs; AC2-AC9). The ability to use this platform to conduct HTS for novel chemical probes that reduce PKA activity was demonstrated by a pilot screen of the LOPAC® 1280 library, leading to the identification of diphenyleneiodonium chloride (DPI) as an inhibitor of basal AC activity. This screening technology could open the door to the development of therapeutic compounds that target GNAS or the ACs, an area in which there is significant unmet need.
(Copyright © 2019 Elsevier Inc. All rights reserved.)
Databáze: MEDLINE