title: Identification of inhibitors using a cell-based assay for monitoring Golgi-resident protease activity.
abstract: Noninvasive real-time quantification of cellular protease activity allows monitoring of enzymatic activity and identification of activity modulators within the protease's natural milieu. We developed a protease activity assay based on differential localization of a recombinant reporter consisting of a Golgi retention signal and a protease cleavage sequence fused to alkaline phosphatase (AP). When expressed in mammalian cells, this protein localizes to Golgi bodies and, on protease-mediated cleavage, AP translocates to the extracellular medium where its activity is measured. We used this system to monitor the Golgi-associated protease furin, a pluripotent enzyme with a key role in tumorigenesis, viral propagation of avian influenza, ebola, and HIV as well as in activation of anthrax, pseudomonas, and diphtheria toxins. This technology was adapted for high-throughput screening of 39,000-compound small molecule libraries, leading to identification of furin inhibitors. Furthermore, this strategy was used to identify inhibitors of another Golgi protease, the beta-site amyloid precursor protein (APP)-cleaving enzyme (BACE). BACE cleavage of the APP leads to formation of the Abeta peptide, a key event that leads to Alzheimer's disease. In conclusion, we describe a customizable noninvasive technology for real-time assessment of Golgi protease activity used to identify inhibitors of furin and BACE.
authors.affiliation: Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor, MI 48109, USA.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Coppola
authors.forename: Christin A
authors.initials: CA
authors.lastname: Hamilton
authors.forename: Mahaveer S
authors.initials: MS
authors.lastname: Bhojani
authors.forename: Martha J
authors.initials: MJ
authors.lastname: Larsen
authors.forename: Brian D
authors.initials: BD
authors.lastname: Ross
authors.forename: Alnawaz
authors.initials: A
authors.lastname: Rehemtulla
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001681:Biological Assay; D002478:Cells, Cultured; D004353:Drug Evaluation, Preclinical / Q000295:instrumentation / Q000379:methods; D005240:Feasibility Studies; D045683:Furin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D006056:Golgi Apparatus / Q000201:enzymology*; D006801:Humans; D007202:Indicators and Reagents / Q000378:metabolism; D011480:Protease Inhibitors / Q000032:analysis* / Q000378:metabolism*; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: D007202:Indicators and Reagents; D011480:Protease Inhibitors; D000469:Alkaline Phosphatase; D053829:Amyloid Precursor Protein Secretases; D045683:Furin