Functional imaging of proteases: recent advances in the design and application of substrate-based and activity-based probes
Highlights
► Explanation of the need for tools to image protease activity. ► Examples of recently developed substrate-based imaging probes for cysteine proteases. ► Examples of recently developed activity-based imaging probes for cysteine proteases. ► Discussion of the pros and cons of substrate and activity based probes.
Introduction
The protease family contains approximately 560 members, comprising nearly 2% of the human genome. The primary function of this diverse family of enzymes is to cleave specific peptide bonds of substrates. While this activity is important for normal cellular processes, it is also a critical regulatory mechanism for many pathologies including cancer, arthritis, atherosclerosis, and neurodegenerative disorders such as Alzheimer's and Huntington's Disease, among others. Proteases are classified into seven subfamilies, according to their mechanism of catalysis. Cysteine, serine, and threonine proteases use a nucleophilic amino acid side chain to catalyze the hydrolysis of the peptide substrate (Figure 1). Metallo and aspartic proteases, on the contrary, use active site residues to deprotonate a water molecule for substrate attack.
Because unchecked proteolysis would be highly detrimental to the cell, proteases are subject to tight regulatory mechanisms. They are synthesized as inactive zymogens that can be activated by a number of mechanisms. Once activated, proteases are often negatively regulated by endogenous protein-based inhibitors. Therefore, to obtain a clear understanding of both the normal and pathological function of proteases, direct assessment of the regulation of their enzymatic activities is required. Traditional tools, such as antibodies or proteomic methods survey total protein levels and therefore do not provide information on the dynamic regulation of protease activity. For this reason, new biochemical tools to study protease activity have been in high demand. This review will primarily discuss two major classes of probes, substrate-based and activity-based probes, and how these reagents have been applied to study the biological function of cysteine proteases biochemically and using optical imaging methods. We aim to provide a crucial interpretation of the pros and cons of each type of probe and to provide insight regarding the future of this technology.
Section snippets
Substrate-based probes
Although proteases were originally thought to completely degrade proteins in order to maintain homeostasis of protein levels in the cell, it is now clear that they perform limited proteolysis of substrates at defined cleavage sites. This allows proteases to regulate structure, function, and localization of substrates. Although the ability to cleave a specific site on a protein substrate can be controlled by a number of factors including tertiary structure and localization of target and
Activity-based probes
As an alternative to substrate-based probes, it is also possible to monitor protease activity using activity-based probes (ABPs). ABPs are compounds that have been engineered to covalently modify enzyme targets in an activity dependent manner. ABPs typically contain a reactive functional group (often referred to as a warhead) linked to a targeting sequence and a tag for visualization or affinity purification (Figure 3A). ABPs have been developed for a number of enzyme families, including
Cathepsins
Papain-like cysteine proteases, or cysteine cathepsins, were once thought to degrade proteins nonspecifically in the lysosome. However, their roles in normal cellular processes and disease pathologies have become increasingly apparent. Cysteine cathepsins are implicated in cancer progression, owing to their roles in angiogenesis, apoptosis, and tumor cell invasion [20]. They are also key regulators of inflammation in diseases such as atherosclerosis, rheumatoid arthritis, and asthma. A number
Pros and cons of substrate-based and activity-based probes
One of the potential drawbacks of the quenched activity-based probes is that, owing to their covalent nature, there is a one-to-one reaction between probe and enzyme, preventing signal amplification. Because the substrate-based probes do not render the protease inactive, one molecule of enzyme is theoretically able to cleave many probe molecules, potentially leading to enhanced fluorescent signal. In order to assess the potential contribution of signal amplification, Blum et al. performed a
Conclusions
Cysteine proteases have been targeted by a significant number of probes, resulting in a wealth of tools that can be used to monitor their activity. Here we have described key advances in the development of fluorescent probes for imaging cysteine proteases. We have highlighted some of the key differences between substrate-based and activity-based probes, providing insight into the strengths and weaknesses of each. Both classes of probes have utility across a wide range of applications, allowing
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
The authors thank Edgar Deu for helpful discussions of the manuscript. This work was funded by National Institutes of Health grants R01 AI-078947 and R01 EB005011 (both to M.B.).
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