266 © 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Biotechnol. J. 2014, 9, 266–281 DOI 10.1002/biot.201300201 www.biotechnology-journal.com Biotechnology Journal 1 Introduction Proteinases are integral to many physiological functions, including digestion, remodeling of the extracellular matrix (ECM), cell migration, induction of apoptosis, trim- ming of cell surface proteins, intracellular signal trans- duction, virus maturation, and defense against patho- gens. Many proteinases act on specific substrates, but uncontrolled and prolonged proteinase activity are likely to lead to tissue damage, as in chronic inflammation of the lung [1, 2]. A good example of the importance of pro- teinases in diseases can be observed in chronic obstruc- tive pulmonary disease (COPD), where the release of elas- tases from white blood cells into the lumen of the airways during chronic inflammation ultimately leads to pul- Review FRET-based and other fluorescent proteinase probes Hai-Yu Hu 1,2, *, Stefanie Gehrig 1,3,4, *, Gregor Reither 1,3,4 , Devaraj Subramanian 1 , Marcus A. Mall 3,4,5,6 , Oliver Plettenburg 2 and Carsten Schultz 1,3,4 1 European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Heidelberg, Germany 2 Sanofi Deutschland GmbH, Diabetes Division, R&D, Industriepark Hoechst, Frankfurt am Main, Germany 3 Translational Lung Research Center (TLRC), Member of the German Center for Lung Research (DZL), University of Heidelberg, Heidelberg, Germany 4 Molecular Medicine Partnership Unit (MMPU), University of Heidelberg and European Molecular Biology Laboratory, Heidelberg, Germany 5 Division of Pediatric Pulmonology & Allergy and Cystic Fibrosis Center, Departments of Pediatrics, University of Heidelberg, Heidelberg, Germany 6 Department of Translational Pulmonology, University of Heidelberg, Heidelberg, Germany The continuous detection of enzyme activities and their application in medical diagnostics is one of the challenges in the translational sciences. Proteinases represent one of the largest groups of enzymes in the human genome and many diseases are based on malfunctions of proteolytic activ- ity. Fluorescent sensors may shed light on regular and irregular proteinase activity in vitro and in vivo and provide a deeper insight into the function of these enzymes and their role in pathophys- iological processes. The focus of this review is on Förster resonance energy transfer (FRET)-based proteinase sensors and reporters because these probes are most likely to provide quantitative data. The medical relevance of proteinases are discussed using lung diseases as a prominent example. Probe design and probe targeting are described and fluorescent probe development for disease-relevant proteinases, including matrix-metalloproteinases, cathepsins, caspases, and oth- er selected proteinases, is reviewed. Keywords: Activity-based probes · Cathepsins · Cell targeting · Chronic inflammatory lung disease · Matrix metalloproteinases Correspondence: Dr. Carsten Schultz, European Molecular Biology Laboratory (EMBL), Cell Biology and Biophysics Unit, Meyerhofstr. 1, 69117 Heidelberg, Germany E-mail: schultz@embl.de Current address: Devaraj Subramanian, SiChem GmbH, Bremen, Germany Abbreviations: ABP, activity-based probes; ACPP, activable cell-penetrating probes; ADAM, a disintegrin-like and metalloproteinase-like proteinase; ADAMTS, a disintegrin-like and metalloproteinase-like proteinase and thrombospondin type 1 motif; CF, cystic fibrosis; CFP, cyan fluorescent pro- tein; COPD, chronic obstructive pulmonary disease; ECM, extracellular matrix; FAP, fibroblast activation protein; FP, fluorescent protein; FRET, Förster resonance energy transfer; MMP, matrix metalloproteinase; NE, neutrophil elastase; PEG, polyethylene glycol; TNF, tumor necrosis factor; YFP, yellow fluorescent protein Biotechnology Journal Received 11 AUG 2013 Revised 25 OCT 2013 Accepted 24 DEC 2013 * These authors contributed equally to this work.