Article

Functional imaging of proteases: Recent advances in the design and application of substrate-based and activity-based probes

Cancer Biology Program, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5324, USA.
Current opinion in chemical biology (Impact Factor: 6.81). 11/2011; 15(6):798-805. DOI: 10.1016/j.cbpa.2011.10.012
Source: PubMed

ABSTRACT

Proteases are enzymes that cleave peptide bonds in protein substrates. This process can be important for regulated turnover of a target protein but it can also produce protein fragments that then perform other functions. Because the last few decades of protease research have confirmed that proteolysis is an essential regulatory process in both normal physiology and in multiple disease-associated conditions, there has been an increasing interest in developing methods to image protease activity. Proteases are also considered to be one of the few 'druggable' classes of proteins and therefore a large number of small molecule based inhibitors of proteases have been reported. These compounds serve as a starting point for the design of probes that can be used to target active proteases for imaging applications. Currently, several classes of fluorescent probes have been developed to visualize protease activity in live cells and even whole organisms. The two primary classes of protease probes make use of either peptide/protein substrates or covalent inhibitors that produce a fluorescent signal when bound to an active protease target. This review outlines some of the most recent advances in the design of imaging probes for proteases. In particular, it highlights the strengths and weaknesses of both substrate-based and activity-based probes and their applications for imaging cysteine proteases that are important biomarkers for multiple human diseases.

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Available from: Laura E Edgington-Mitchell, Mar 02, 2014
    • "While clinical trials with inhibitors of MMPs were ill-defined and entered the clinical phase prematurely, the example of bortezomib as a proteasome inhibitor that blocks cellular protein turnover, and therefore acts in a much less specific manner, has been used successfully to treat certain tumors. In addition to the usefulness of protease inhibitors to study protease function in vitro, activity-based probes have proven to be extremely useful to spot protease activities in situ (Blum 2008;Edgington et al. 2011;Nomura et al. 2010;Puri and Bogyo 2013;Salpeter and Blum 2013;Shen 2012). Now, we have reached the level of theranostics (Lee and Kim 2012;Mikhaylov et al. 2014), which comprises therapeutic molecules that are targeted to a desired site of action relying on antibody-based immunogenic recognition of the target cell or tissue. "
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    ABSTRACT: Proteases play essential roles in protein degradation, protein processing, and extracellular matrix remodeling in all cell types and tissues. They are also involved in protein turnover for maintenance of homeostasis and protein activation or inactivation for cell signaling. Proteases range in function and specificity, with some performing distinct substrate cleavages, while others accomplish proteolysis of a wide range of substrates. As such, different cell types use specialized molecular mechanisms to regulate the localization of proteases and their function within the compartments to which they are destined. Here, we focus on the cysteine family of cathepsin proteases and legumain, which act predominately within the endo-lysosomal pathway. In particular, recent knowledge on cysteine cathepsins and their primary regulator legumain is scrutinized in terms of their trafficking to endo-lysosomal compartments and other less recognized cellular locations. We further explore the mechanisms that regulate these processes and point to pathological cases which arise from detours taken by these proteases. Moreover, the emerging biological roles of specific forms and variants of cysteine cathepsins and legumain are discussed. These may be decisive, pathogenic, or even deadly when localizing to unusual cellular compartments in their enzymatically active form, because they may exert unexpected effects by alternative substrate cleavage. Hence, we propose future perspectives for addressing the actions of cysteine cathepsins and legumain as well as their specific forms and variants. The increasing knowledge in non-canonical aspects of cysteine cathepsin- and legumain-mediated proteolysis may prove valuable for developing new strategies to utilize these versatile proteases in therapeutic approaches.
    No preview · Article · Nov 2014 · Protoplasma
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    • "Under acidic conditions cathepsins are far more efficient proteases than at neutral pH, partially due to the instability of their substrates, and partially due to their higher stability [13], thereby suggesting that extracellular cathepsins may even have a leading role in the degradation of ECM [83]. A further confirmation of such cathepsin activity was obtained by in vivo imaging approaches using activitybased probes against different cathepsins [84] [85] [86] "
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    ABSTRACT: Cysteine cathepsins are normally found in the lysosomes where they are involved in intracellular protein turnover. Their ability to degrade the components of the extracellular matrix in vitro was first reported more than 25 years ago. However, cathepsins were for a long time not considered to be among the major players in ECM degradation in vivo. During the last decade it has, however, become evident that abundant secretion of cysteine cathepsins into extracellular milieu is accompanying numerous physiological and disease conditions, enabling the cathepsins to degrade extracellular proteins. In this review we will focus on cysteine cathepsins and their extracellular functions linked with ECM degradation, including regulation of their activity, which is often enhanced by acidification of the extracellular microenvironment, such as found in the bone resorption lacunae or tumor microenvironment. We will further discuss the ECM substrates of cathepsins with a focus on collagen and elastin, including the importance of that for pathologies. Finally, we will overview the current status of cathepsin inhibitors in clinical development for treatment of ECM-linked diseases, in particular osteoporosis. Due to their major role in ECM remodelling cysteine cathepsins have emerged as an important group of therapeutic targets for a number of ECM-related diseases, including, osteoporosis, cancer and cardiovascular diseases. This article is part of a Special Issue entitled Matrix-mediated cell behaviour and properties..
    Full-text · Article · Mar 2014 · Biochimica et Biophysica Acta
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    • "In this study, it is evident that targeting of NE on NETs could minimize adverse effects of unregulated proteolysis associated with tissue damage and inflammation. The selective cleavage sequences of the neutrophil serine proteases identified in this study will be valuable for designing substrates, inhibitors, activity based imaging agents [36] and protease-activated prodrugs [37]. In addition, the substrate signature of NETs-associated protease activity can be monitored as a biomarker for inflammatory diseases driven by extracellular neutrophil proteases. "
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    ABSTRACT: Neutrophil extracellular traps (NETs) consist of antimicrobial molecules embedded in a web of extracellular DNA. Formation of NETs is considered to be a defense mechanism utilized by neutrophils to ensnare and kill invading pathogens, and has been recently termed NETosis. Neutrophils can be stimulated to undergo NETosis ex vivo, and are predicted to contain high levels of serine proteases, such as neutrophil elastase (NE), cathepsin G (CG) and proteinase 3 (PR3). Serine proteases are important effectors of neutrophil-mediated immunity, which function directly by degrading pathogenic virulent factors and indirectly via proteolytic activation or deactivation of cytokines, chemokines and receptors. In this study, we utilized a diverse and unbiased peptide library to detect and profile protease activity associated with NETs induced by phorbol-12-myristate-13-acetate (PMA). We obtained a "proteolytic signature" from NETs derived from healthy donor neutrophils and used proteomics to assist in the identification of the source of this proteolytic activity. In addition, we profiled each neutrophil serine protease and included the newly identified enzyme, neutrophil serine protease 4 (NSP4). Each enzyme had overlapping yet distinct endopeptidase activities and often cleaved at unique sites within the same peptide substrate. The dominant proteolytic activity in NETs was attributed to NE; however, cleavage sites corresponding to CG and PR3 activity were evident. When NE was immunodepleted, the remaining activity was attributed to CG and to a lesser extent PR3 and NSP4. Our results suggest that blocking NE activity would abrogate the major protease activity associated with NETs. In addition, the newly identified substrate specificity signatures will guide the design of more specific probes and inhibitors that target NET-associated proteases.
    Full-text · Article · Sep 2013 · PLoS ONE
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