title: A tunable, modular approach to fluorescent protease-activated reporters.
abstract: Proteases are one of the most important and historically utilized classes of drug targets. To effectively interrogate this class of proteins, which encodes nearly 2% of the human proteome, it is necessary to develop effective and cost-efficient methods that report on their activity both in vitro and in vivo. We have developed a robust reporter of caspase proteolytic activity, called caspase-activatable green fluorescent protein (CA-GFP). The caspases play central roles in homeostatic regulation, as they execute programmed cell death, and in drug design, as caspases are involved in diseases ranging from cancer to neurodegeneration. CA-GFP is a genetically encoded dark-to-bright fluorescent reporter of caspase activity in in vitro, cell-based, and animal systems. Based on the CA-GFP platform, we developed reporters that can discriminate the activities of caspase-6 and -7, two highly related proteases. A second series of reporters, activated by human rhinovirus 3C protease, demonstrated that we could alter the specificity of the reporter by reengineering the protease recognition sequence. Finally, we took advantage of the spectrum of known fluorescent proteins to generate green, yellow, cyan, and red reporters, paving the way for multiplex protease monitoring.
authors.affiliation: Department of Chemistry, University of Massachusetts Amherst, Amherst, Massachusetts, USA.
authors.forename: Peng
authors.initials: P
authors.lastname: Wu
authors.forename: Samantha B
authors.initials: SB
authors.lastname: Nicholls
authors.forename: Jeanne A
authors.initials: JA
authors.lastname: Hardy
mesh_terms: D020169:Caspases / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D059748:Proteolysis
chemical_list: D015335:Molecular Probes; D049452:Green Fluorescent Proteins; D020169:Caspases
references.citation: Science. 2009 May 8;324(5928):804-7
references.pmid: 19423828
references.citation: J Biotechnol. 2008 Nov 6;138(1-2):17-23
references.pmid: 18775457
references.citation: Protein Sci. 2013 Mar;22(3):247-57
references.pmid: 23139158
references.citation: Nucleic Acids Res. 2012 Jan;40(Database issue):D343-50
references.pmid: 22086950
references.citation: Cell. 2006 Jun 16;125(6):1179-91
references.pmid: 16777606
references.citation: Protein Sci. 2011 Aug;20(8):1421-31
references.pmid: 21674661
references.citation: Biochem J. 2009 Oct 12;423(3):429-39
references.pmid: 19694615
references.citation: J Biol Chem. 1990 Jun 5;265(16):9062-5
references.pmid: 2160953
references.citation: J Cell Biol. 2003 Jan 20;160(2):235-43
references.pmid: 12527749
references.citation: Chem Biol. 2009 Nov 25;16(11):1169-79
references.pmid: 19942140
references.citation: BMC Biotechnol. 2009 Mar 25;9:24
references.pmid: 19321010
references.citation: Curr Protein Pept Sci. 2007 Feb;8(1):19-27
references.pmid: 17305557
references.citation: J Mol Biol. 2011 Feb 11;406(1):75-91
references.pmid: 21111746
references.citation: Clin Cancer Res. 2008 Sep 15;14(18):5801-9
references.pmid: 18794090
references.citation: J Biol Chem. 2011 Jul 15;286(28):24977-86
references.pmid: 21558267
references.citation: Nat Methods. 2005 Dec;2(12):905-9
references.pmid: 16299475
references.citation: Nat Rev Drug Discov. 2006 Sep;5(9):785-99
references.pmid: 16955069
references.citation: Biochem J. 2000 Sep 1;350 Pt 2:563-8
references.pmid: 10947972
references.citation: Chem Soc Rev. 2009 Oct;38(10):2951-65
references.pmid: 19771338
references.citation: J Biol Chem. 2001 Aug 3;276(31):29188-94
references.pmid: 11387331
references.citation: J Mol Biol. 2008 Mar 21;377(2):565-74
references.pmid: 18258258
references.citation: Nat Biotechnol. 2002 Jan;20(1):87-90
references.pmid: 11753368
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 16;105(37):13901-5
references.pmid: 18779587
references.citation: Nat Methods. 2007 Sep;4(9):741-6
references.pmid: 17721542
references.citation: EMBO Rep. 2010 Nov;11(11):841-7
references.pmid: 20890311
references.citation: Nat Biotechnol. 2004 Apr;22(4):445-9
references.pmid: 14990965
references.citation: J Cell Biol. 2011 Dec 12;195(6):1047-60
references.pmid: 22162136
references.citation: Nat Struct Mol Biol. 2012 Jan 05;19(1):9-16
references.pmid: 22218294
references.citation: Mol Cell Proteomics. 2009 Dec;8(12):2700-14
references.pmid: 19759058
references.citation: Biotechniques. 2002 Dec;33(6):1284-7, 1289-91
references.pmid: 12503313
references.citation: Proc Natl Acad Sci U S A. 2006 May 2;103(18):7130-5
references.pmid: 16641106
references.citation: Curr Biol. 1996 Feb 1;6(2):178-82
references.pmid: 8673464
references.citation: J Biol Chem. 1997 Nov 7;272(45):28545-9
references.pmid: 9353317
references.citation: Proc Natl Acad Sci U S A. 2012 Apr 10;109(15):5669-74
references.pmid: 22451931